Annex B 

Spirotetramat 

B.5 Methods of analysis 

Reviewed by 

PMRA, Canada 

Peer – reviewed by: 

US-EPA, USA 

AGES, Austria 
– 2 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE OF CONTENTS 

B.5.1 Analytical methods for technical active substance and formulation analysis 

(Annex IIA 4.2, Annex IIIA 5.2) ...............................................................................................3

B.5.1.1 Methods for the analysis of pure active substance in the active substance as 

manufactured (Annex II 4.2.1) ..................................................................................................3

B.5.1.2 Applicability of existing CIPAC methods (Annex IIA 4.2.2) ......................................................4

B.5.1.3 Description of analytical methods for the determination of impurities (Annex IIA 4.2.3)..........5

B.5.1.4 Description of analytical methods for the determination of additive (Annex IIA 4.2.4).............5

B.5.1.5 Enforcement analytical methodology (Annex IIA 4.2.5)............................................................5

B.5.1.6 Inter-Laboratory analytical methodology validaton (Annex IIA 4.2.6).......................................5

B.5.1.7 Storage stability of working solutions in analytical methodology (Annex IIA 4.2.6)..................5

B.5.1.8 Analytical methods for the determination of the active substance in plant protection 

products (Annex IIIA 5.2) ..........................................................................................................6

B.5.1.9 Applicability of existing CIPAC methods (Annex IIIA 5.2.3) ..................................................11

B.5.1.10 Analytical methods for the determination of relevant impurities which are of 

toxicological, ecotoxicological or environmetal concern, in the preparation (Annex IIIA 

5.2.4).......................................................................................................................................11

B.5.1.11 Analytical methods for formulants or their constituents in the plant protection product. 

(Annex IIIA 5.2.5)....................................................................................................................11

B.5.2 Analytical methods for the determination of residues (all components included 

in the residue definition proposed (Annex IIA 4.3, Annex IIIA 5.3)..................................12

B.5.2.1 Plant material..........................................................................................................................12

B.5.2.2 Foodstuff of animal origin, feeding stuffs................................................................................24

B.5.3 Analytical methods for the determination of residues in soil, water, sediment 

and air (Annex IIA 4.4 to 4.7, Annex IIIA 5.4 to 5.7) ...........................................................33

B.5.3.1 Soil (Annex IIA 4.4, Annex IIIA 5.4) ........................................................................................33

B.5.3.2 Water (Annex IIA 4.5, Annex IIIA 5.5).....................................................................................39

B.5.3.3 Sediment (Annex IIA 4.6, Annex IIIA 5.6)...............................................................................44

B.5.3.4 Air (Annex IIA 4,7, Annex IIIA 5.7)).........................................................................................44

B.5.4 Analytical methods for the determination or residues in body fluids and tissues 

(Annex IIA 4,8; Annex IIIA 5.8).............................................................................................47

B.5.5 Evaluation and assessment.................................................................................................48

B.5.5.1 Analytical methods for technical active substance and formulation analysis .........................48

B.5.5.2 Analytical methods for residues..............................................................................................48

B.5.6 References relied on ............................................................................................................51
– 3 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.1 Analytical methods for technical active substance and formulation analysis (Annex IIA 

4.2, Annex IIIA 5.2) 

B.5.1.1 Methods for the analysis of pure active substance in the active substance as 

manufactured (Annex II 4.2.1) 

Report: KIIA 4.2.1/01, Hake, 2004 

Title: Spirotetramat (BYI 08330) - Assay of Technical Grade Active Ingredient - 

HPLC - External Standard 

Report No & 

Document No 

AM001104MP1 

M-084385-01-1 

Dates of work: 2004-07-07 

GLP Non GLP 

Report: KIIA 4.2.1/02, Hake, 2004 

Title: Validation of HPLC-method AM001104MP1 - BYI 08330 Assay of Technical Grade 

Active Ingredient 

Report No & 

Document No 

VB1-AM001104MP1 

M-084403-01-1 

Dates of work: 2004-07-07 

GLP Non GLP 

The HPLC- method AM001104MP1 is used for the determination of spirotetramat in technical grade active 

substance. 

Principle of the method: 

The components are separated by reverse phase high performance liquid chromatography (HPLC). The 

quantitative evaluation is carried out using the peak area of spirotetramat by comparison with the area of the 

calibration substance according to the method of external standardization. The following table presents the 

details of method AM001104MP1. 
– 4 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Method ID AM001104MP1 

Analyte Active 

Sample preparation Homogenize thoroughly approx. 5 g of the sample in an agate mortar. 

Transfer approx. 100 mg of this material accurately weighed to 0.00001 g to 

a 100 mL flask and make up to mark with acetonitrile. 

Instrument HPLC 

Detector UV at 230 nm 

Column Phenomenex Luna C18, 200 mm × 4.6 mm, 3 μm 

Mobile phase Gradient of A and B where: 

A = 0.1% aqueous phosphoric acid 

B = methanol 

Time (min) % A % B Flow rate (mL/min) 

0 

36 

39 

41 

45 

45 

45 

5 

5 

45 

55 

55 

95 

95 

55 

1.2 

1.2 

1.2 

1.2 

1.2 

Oven temperature 43 °C 

Quantitation External standard 

Total run time 36 minutes 

Retention time Spirotetramat 31.0 minutes 

The HPLC method AM001104MP1 for the determination of spirotetramat in technical grade active substance 

has been completely validated by checking the parameters linearity, precision, accuracy, specificity and 

interference. 

Linearity 6 concentrations with double measurements; range 78 - 123 %; 

correlation coefficient rK: 0.9992; 

regression equation: y = 64.674x + 33.631. 

Chromatograms are given; the function is linear in the operating range. 

Precision 

Repeatability 

6 synthetic samples are measured; 

no outliers are reported, RSD: 0.50 %; 

acceptable according to the Horwitz equation. 

Accuracy 6 synthetic samples are measured; 

mean recovery: 100.3 %; RSD: 0.50 %. 

Specificity/Interference The UV-spectra of reference substance and analyte in the sample show 

no spectral difference; the retention times are identical. 

No interferences are found. 

Conclusion: The analytical method provided was fully validated and assessed to be precise, selective and 

accurate for the determination of the active ingredient spirotetramat. 

B.5.1.2 Applicability of existing CIPAC methods (Annex IIA 4.2.2) 

Up to now there is no CIPAC method available for the determination of spirotetramat in technical grade 

active substance. 
– 5 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.1.3 Description of analytical methods for the determination of impurities (Annex IIA 4.2.3) 

Description in volume 4, confidential part 

B.5.1.4 Description of analytical methods for the determination of additive (Annex IIA 4.2.4) 

Refer to file of confidential information and data provided separately. 

B.5.1.5 Enforcement analytical methodology (Annex IIA 4.2.5) 

Not required for the methods for the analysis of the active substance as manufactured. 

B.5.1.6 Inter-Laboratory analytical methodology validaton (Annex IIA 4.2.6) 

Not required for the methods for the analysis of the active substance as manufactured. 

B.5.1.7 Storage stability of working solutions in analytical methodology (Annex IIA 4.2.6) 

Not required for the methods for the analysis of the active substance as manufactured. 
– 6 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.1.8 Analytical methods for the determination of the active substance in plant protection 

products (Annex IIIA 5.2) 

Analytical method for the determination of Spirotetramat in Movento® 240 SC Insecticide 

Report: KIIIA1 5.2/01, Zitzmann; 2004 

Title: Determination of BYI 08330 in Formulations; Assay – HPLC – External Standard 

Report No & 

Document No 

AM002104MF1 

M-068817-01-2 

Dates of work 2004-05-11 

GLP Non GLP 

Report: KIIIA1 5.2/02, Odendahl; 2004 

Title: Validation of HPLC-method AM002104MF1 -Determination of BYI 08330 in 

FormulationsReport No & 

Document No 

VB1-AM002104MF1 

M-068807-01-1 

Dates of work 2004-05-05 

GLP Non GLP 

Report: KIIIA1 5.2/03, Odendahl; 2006 

Title: Validation of HPLC-method AM002104MF1 -Determination of Spirotetramat in 

FormulationsReport No & 

Document No 

VS3.1-AM002104MF1 

M-269319-01-1 

Dates of work 2006-04-07 

GLP Non GLP 

The HPLC method AM002104MF1 is applicable for the determination of the content of spirotetramat in 

formulations (e.g. ‘Spirotetramat 240 g/L SC’). 

Principle of the method: 

The components are separated by reversed phase chromatography using gradient elution (1L water + 5 ml 

1N sulphuric acid / acetonitrile, 52/48 (v/v)). After UV detection (240 nm), the quantitative evaluation is 

carried out by comparing the peak areas with those of reference substances, using an external standard. 

The following table presents the details of the method. 
– 7 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Method ID AM002104MF1 

Sample preparation Transfer an amount of the homogeneous sample containing approx. 50 mg BYI 

08330, accurately weighed to 0.1 mg, into a 100 mL volumetric flask. Add 5 mL of 

water to suspend the sample and then dissolve in approx. 50 mL acetonitrile. To 

dissolve the substance, place the flask in an ultrasonic bath for 15 min. Fill up the 

flask with water to just below the calibration mark and mix. Wait until the 

temperature has stabilized, then fill the flask up to the calibration mark with water. 

Instrument HPLC 

Detector UV at 240 nm 

Column Prism RP 8, 5 mm*

× 4.6 mm, 3 μm 

Mobile phase Gradient of A and B where: 

A = 1 liter water + 5 mL 1N sulphuric acid 

B = acetonitrile 

Time (min) % A % B Flow rate (mL/min) 

0 

1.5 

1.51 

2.2 

2.21 

2.6 

52 

52 

5 

5 

52 

52 

48 

48 

95 

95 

48 

48 

2 

2 

3 

3 

3 

2 

Oven temperature 50 °C 

Quantitation External standard 

Retention time 1.2 min 

Total run time 3 minutes 

The HPLC method AM002104MF1 has been completely validated on the formulation ‘Spirotetramat 240 g/L 

SC (internal code: 0329757)’ by checking the parameters linearity, precision, accuracy, specificity and 

interference from excipients. Additionally interference from excipients is checked on the formulation 

‘Spirotetramat 240 g/L SC (internal code: 06424384)’. 
– 8 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Linearity 6 concentrations with single measurements; range 50 -150 %; correlation 

co-efficient rK: 1.0000; 

regression equation: y = 0.9968x + 0.3408. 

Chromatograms are given; 

the function is linear and proportional in the operating range. 

Precision 6 samples from one batch; single injection; 

no outliers; acceptable according to the Horwitz equation; 

relative standard deviation CV: 0.25 %. 

Accuracy 6 samples of laboratory-prepared synthetic formulation; 

recovery: 100.29 %, RSD: 0.48 %; 

the method shows no constant or proportional systematic error. 

Specificity The UV-spectra of active substance and reference substance show no 

spectral difference; the retention times of active substance and reference 

substance are identical. 

Interference No interferences are found. 

Conclusion: 

An analytical method was provided for the determination of the active. The method was assessed to be 

selective, precise and accurate for use as an enforcement analytical method. However, the applicant must 

clarify what is the length of the HPLC column used.

The following deficiencies are identified and remain to be addressed. 

Title: Enforcement Analytical Method 

Deficiencies: The length of the HPLC column used was stated as 5 mm which is incorrect. 

Clarification required: The applicant is requested to clarify the length of the HPLC column used. 
– 9 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Analytical method for the determination of Spirotetramat in Movento® 150 OD Insecticide. 

Report: KIIIA1 5.2/01, Zitzmann; 2004 

Title: Determination of BYI 08330 in Formulations; Assay – HPLC – External Standard 

Report No & 

Document No 

AM002104MF1 

M-068817-01-2 

Dates of work 2004-05-11 

GLP Non GLP 

Report: KIIIA1 5.2/02, Odendahl; 2005 

Title: Validation of HPLC-method AM002104MF1 -Determination of BYI 08330 in 

FormulationsReport No & 

Document No 

VB2-AM002104MF1 

M-260838-01-1 

Dates of work 2005-11-14 

GLP Non GLP 

The HPLC method AM002104MF1 is applicable for the determination of the content of spirotetramat in 

formulations (e.g. ‘Spirotetramat 150 g/L OD’). 

Principle of the method: 

The components are separated by reversed phase chromatography using gradient elution (1L water + 5 ml 

1N sulphuric acid / acetonitrile, 52/48 (v/v)). After UV detection (240 nm), the quantitative evaluation is 

carried out by comparing the peak areas with those of reference substances, using an external standard. 

The following table presents the details of the method. 

Method ID AM002104MF1 

Sample preparation Transfer an amount of the homogeneous sample containing approx. 50 mg BYI 

08330, accurately weighed to 0.1 mg, into a 100 mL volumetric flask and dissolve in 

approx. 80 - 90 mL of solvent mixture of acetonitrile / tetrahydrofuran / 2-propanol, 

2/3/5 and place the flask in an ultrasonic bath for 15 min. Fill up the flask with water 

to just below the calibration mark and mix. Wait until the temperature has 

stabilized, then fill the flask up to the calibration mark with water. 

Instrument HPLC 

Detector UV at 240 nm 

Column Prism RP 8, 5 mm*

× 4.6 mm, 3 μm 

Mobile phase Gradient A = 1 liter water + 5 mL 1N sulphuric acid 

Gradient B = acetonitrile 

Time (min) % A % B Flow rate (mL/min) 

0 

1.5 

1.51 

2.2 

2.21 

2.6 

52 

52 

5 

5 

52 

52 

48 

48 

95 

95 

48 

48 

2 

2 

3 

3 

3 

2 

Oven temperature 50 °C 
– 10 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Quantitation External standard 

Retention time 1.2 min 

Total run time 3 minutes 

The HPLC method AM002104MF1 has been completely validated on the formulation 

‘Spirotetramat 150 g/L OD’ by checking the parameters linearity, precision, accuracy, specificity and 

interference from excipients. 

Linearity 6 concentrations with single measurements; range 50 -150 %; correlation 

co-efficient rK: 1.0000; 

regression equation: y = 0.9968x + 0.3408. 

Chromatograms are given; 

the function is linear and proportional in the operating range. 

Precision 6 samples from one batch; single injection; 

no outliers; acceptable according to the Horwitz equation; 

relative standard deviation CV: 0.50 %. 

Accuracy 6 samples of laboratory-prepared synthetic formulation; 

recovery: 100.29 %, RSD: 0.48 %; 

the method shows no constant or proportional systematic error. 

Specificity The UV-spectra of active substance and reference substance show no 

spectral difference; the retention times of active substance and reference 

substance are identical. 

Interference No interferences are found. 

Conclusion: 

An analytical method was provided for the determination of the active. The method was assessed to be 

selective, precise and accurate for use as an enforcement analytical method. However, the applicant must 

clarify what is the length of the HPLC column used.
– 11 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.1.9 Applicability of existing CIPAC methods (Annex IIIA 5.2.3) 

A CIPAC method for the determination of spirotetramat in formulations is not available. 

B.5.1.10 Analytical methods for the determination of relevant impurities which are of 

toxicological, ecotoxicological or environmental concern, in the preparation (Annex 

IIIA 5.2.4) 

Neither Movento® 240 SC Insecticide nor Movento® 150 OD Insecticide contains any impurities which are of 

toxicological, ecotoxicological or environmental concern. 

B.5.1.11 Analytical methods for formulants or their constituents in the plant protection product 

(Annex IIIA 5.2.5) 

With respect to toxicological, ecotoxicological or environmental aspects neither Movento® 240 SC 

Insecticide nor Movento® 150 OD Insecticide contains formulants in relevant concentrations. Therefore a 

special analytical method and validation is not needed. 
– 12 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.2 Analytical methods for the determination of residues (all components included in the 

residue definition proposed (Annex IIA 4.3, Annex IIIA 5.3) 

B.5.2.1 Plant material 

Report: KIIA 4.3/01, Schöning, R., Stuke, S., Billian, P.; 2005 

Title: Analytical method 00857 for the determination of residues of BYI08330 (parent 

compound and total residue), BYI08330-enol, BYI08330-ketohydroxy, BYI08330-

mono-hydroxy and BYI08330-enol-Glc metabolite in/on plant material by HPLCMS/MS 

Report No & 

Edition No 

MR-099/04 including Report MR-148/04: (Schoening, R, 2005) Supplement E001 of 

the analytical method 00857 for the determination of residues of BYI08330 (parent 

compound and total residue), BYI08330-enol, BYI08330-ketohydroxy, BYI08330-

mono-hydroxy and BYI08330-enol-GLC metabolite in/on plant material by HPLCMS/MS 

M-253112-02-1

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

EU Guidance document for generating and reporting Methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP yes 

Report: KIIA 4.3/02, Freitag, T.; Daniels, M.; 2005 

Title: Analytical Method 00857/M003 for the determination of residues of BYI08330 and 

BYI08330-ketohydroxy metabolite in/on plant material by HPLC-MS/MS 

Report No & 

Edition No 

MR-098/05 

M-263485-01-2 

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

EU Guidance document for generating and reporting Methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP yes 

Report: KIIA 4.3/03, Spiegel, K.; 2006 

Title: [Azaspirodecenyl-2-14C]BYI08330: Extraction efficiency testing of the residue 

method for the determination of BYI08330, BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-monohydroxy and BYI08330-enol glucoside in cotton gin trash, in apple 

fruit and in lettuce using aged radioactive residues 

Report No & 

Edition No 

MEF-05/022 

M-248725-01-2 

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340. 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/04, Brookey, FM; 2006 

Title: Independent laboratory validation of the residue analytical method: “Analytical 

method 00857 for the determination of residues of BYI08330 (parent compound and 

total residue of BYI08330), BYI08330-enol, BYI08330-ketohydroxy, BYI08330-

monohydroxy and BYI08330-enol-Glc metabolite in/on plant material by HPLCMS/MS” 

Report No & 

Edition No 

RAFNP008 

M-277335-01-1 
– 13 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340. 

EU-Guidelines SANCO/825/00 rev.7 Guidance Document on Residue 

Analytical Methods 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/05, Pruitt WE; 2006 

Title: FDA PAM Multiresidue Method (MRM) 

Report No & 

Edition No 

RAFNP007 

M-277248-01-1 

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 and OPPTTS 

860.1360 Multiresidue Method Testing 

PMRA Regulatory Directive 98-02; DACO 7.2.4 

GLP Yes 

Report: KIIA 4.3/18, Schöning, R., Willmes, J., 2008 

Title: Analytical method 01084 for the determination of residues of spirotetramat 

(BYI 08330), BYI08330-enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and 

BYI08330-enol-glucoside metabolites in/on plant material by HPLC-MS/MS 

Report No & 

Edition No 

MR-08/007 

M-298287-01-1 

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

EU Guidance Document for generating and reporting methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 
– 14 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

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

EU Guidance Document on residue analytical methods SANCO/3029/99 rev.7 

of March 19, 2004 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/08, Klimmek, S.; 2005 

Title: BYI08330-enol: examination of the applicability of DFG method S 19 (extended and 

revised revision) for the determination of residues of BYI08330-enol 

Report No & 

Edition No 

BAY-0519 Az. G05-0104 

M-257512-01-1 

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

EU Guidance Document SANCO/825/00 

BBA-guideline: Residue analytical methods for post-registration control 

purposes of July 21, 1998 

GLP Yes 

Report: KIIA 4.3/09, Freitag, T.; 2005 amended 02.06.2006 

Title: Analytical method 00929 for the determination of residues of BYI08330-

ketohydroxy-alcohol, BYI08330-desmethyl-ketohydroxy and BYI08330-desmethyldi-hydroxy in/on plant material by HPLC-MS/MS 

Report No & 

Edition No 

MR-026/05 

M-256183-01-2 

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

EU Guidance Document for generating and reporting methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/10, Klempner, A., Spiegel, K; 2006

Title: [Azaspirodecenyl-3-14C] BYI08330: extraction efficiency testing (radiovalidation) of 

the residue method (00929) for the determination of BYI08330-ketohydroxy-alcohol, 

BYI08330-desmethyl-ketohydroxy and BYI08330-desmethyl-di-hydroxy in plant 

samples using aged radioactive residues 

Report No & 

Edition No 

MEF-06/008 

M-270054-02-2 

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 
– 15 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Principle of the Data-Gathering Method:

Analytical method 00857

Spirotetramat and the metabolites BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy and 

BYI 08330-enol-Glc in/on plants were extracted using a blender with an acidic ACN/water mixture (4/1, v/v). 

After filtration and clean-up of the extract, the corresponding stable isotopically labelled analytes were added 

as internal standards. Afterwards, the solution was made up to volume, diluted and subjected to HPLCMS/MS (TABLE B.5.2.1-1). 

TABLE 5.2.1-1. Summary Parameters for the Analytical Method Used for the Quantitation of 

Spirotetramat Residues in Plant Matrices.

Method ID Analytical method 00857 

Analyte(s) Spirotetramat, BYI08330-enol, BYI 08330-ketohydroxy, 

BYI 08330-mono-hydroxy and BYI 08330-enol-Glc 

Extraction solvent/technique acidic ACN/water mixture (4/1, v/v) 

Cleanup strategies filter through BondElut Clean polyethylene frit 

Instrument/Detector Liquid chromatograph Agilent 1100 with MS (API 4000 with ESI interface 

and MS detector) 

Column: Agilent Zorbax Eclipse-XDB-C8, 5 μm, 150x4.6 mm i.d. 

with pre-column or equivalent. 

Mobile phase: 

Pump A: water/ACN (9/1, v/v), containing 0.22 

mL/L formic acid. 

Pump B: ACN, containing 0.22 mL/L formic acid 

Pump C: water/ACN (1/1, v/v), containing 0.1 mL/L acetic acid or 

formic acid 

Ionization: ESI, positive 

1

st MRM Spirotetramat 374 to 216 m/z

 BYI 08330-enol 302 to 216 m/z 

 BYI 08330-ketohydroxy 318 to 268 m/z 

BYI 08330-mono-hydroxy 304 to 254 m/z 

BYI 08330-enol-Glc 464 to 270 m/z

2

nd MRM* Spirotetramat 374 to 302 m/z

(confirmatory) BYI 08330-enol 302 to 270 m/z 

 BYI 08330-ketohydroxy 318 to 214 m/z 

BYI 08330-mono-hydroxy 304 to 119 m/z 

BYI 08330-enol-Glc 464 to 216 m/z

Standardization method Mixed stable isotope internal standards of spirotetramat, BYI 08330-

enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy and BYI 08330-

enol-Glc. 

Stability of std solutions All analytes are stable in standard solutions for at least 4 weeks of 

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

Retention times Spirotetramat (~4.3 minutes), BYI 08330-enol (~2.7 minutes), BYI 

08330-ketohydroxy (~3.2 minutes), BYI 08330-mono-hydroxy (~2.5 

minutes) and BYI 08330-enol-Glc (~1.6 minutes). 
– 16 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.1-2 Characteristics for the Data-Gathering Analytical Method 00857 used for the 

Quantitation of Spirotetramat Residues in Plant Matrices. 

Analyte(s) Spirotetramat, BYI08330-enol, BYI 08330-ketohydroxy, 

BYI 08330-mono-hydroxy, and BYI 08330-enol-Glc 

Equipment ID Agilent 1100 HPLC system 

LOQ 0.01 ppm for each analyte in all plant matrices with the exception of hop 

cones. 0.1 ppm for each analyte in hop cones. 

LOD The LOD for all analytes was estimated to be 4-10 fold lower than the 

corresponding LOQ. 

Accuracy/Precision Range of percent recoveries ± coefficient of variation indicated acceptable 

accuracy/precision in the spiking levels of 0.01 ppm and 1.0 ppm of each 

analyte for plant matrices (except hops), and 0.1-10 ppm in hop cones. 

Mean recoveries of all analytes from all tested matrices were within the 

acceptable range of 70-120% with RSD <16%. 

Reliability of the Method/ [ILV] An independent laboratory method validation [ILV], Study No. RAFNP008, 

was conducted to verify the reliability of method No. 00857 for the 

determination of spirotetramat residues in plant matrices. The values 

obtained are indicative that method No. 00857 is reliable. 

Linearity The method/detector response was linear (r

2

 >0.997) within the range of 5 

ng/L-50 μg/L. 

Specificity The control chromatograms generally have no peaks above the 

chromatographic background and the spiked sample chromatograms 

contain only the analyte peak of interest. Peaks were well defined and 

symmetrical. Two MRM transitions were monitored for each analyte/matrix 

for quantitation and confirmation purposes. 

The extraction efficiency of method 00857 was tested using cotton gin trash, lettuce, and apples containing 

aged radioactive residues from metabolism studies. The recoveries of the extracted radioactive residues of 

spirotetramat and the metabolites in cotton gin trash, lettuce and apple samples using method 00857 were in 

agreement with those generated in the metabolism studies. 

TABLE B.5.2.1-3. Extraction Efficiency of Method 00857 using Radiolabelled Samples from 

Cotton, Lettuce and Apple Metabolism Studies.

Crops Cotton gin trash 

 (% extracted) 

Lettuce 

(% extracted) 

Apple 

 (% extracted) 

 Method 

00857 

Metabolism 

study 

Method 

00857 

Metabolis

m study 

Method 

00857 

Metabolism 

study 

% TRRs extracted 87.4 88.6 95.5 95.8 96.5 98.0 

% of Compounds in the extract 

Spirotetramat 16.8 19.8 48.2 55.9 47.5 51.3 

BYI 08330-enol 13.2 12.1 24.0 17.8 2.9 2.1 

BYI 08330-enol-Glc 3.6 4.0 11.2 11.4 5.3 5.1 

BYI 08330-

ketohydroxy 

31.8 29.7 12.0 6.2 8.7 7.7 

BYI 08330-monohydroxy -- -- -- -- 15.6 15.6 
– 17 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Analytical method 00929 for data-gathering in rotational crops

Principle of the method: 

BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl-ketohydroxy and BYI 08330-desmethyl-di-hydroxy 

and the corresponding conjugates in/on plants were extracted using a blender with ACN/water (1/1, v/v). 

After filtration, the extract was concentrated to the aqueous remainder and hydrolyzed with 3N hydrochloric 

acid, and cleaned up by eluting through the polystyrenedivenylbenzene column. Afterwards, the 

corresponding stable isotopically labelled analytes were added as internal standards, the solution was made 

up to volume and subjected to HPLC-MS/MS (TABLE B.5.2.1-4). 

TABLE B.5.2.1-4. Summary Parameters for the Analytical Method Used for the Quantitation of 

Spirotetramat Residues in Rotational Crop Plant Matrices.

Method ID Analytical method 00929 

Analyte(s) BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl-ketohydroxy and 

BYI 08330-desmethyl-di-hydroxy 

Extraction solvent/technique ACN/water mixture (1/1, v/v) 

Cleanup strategies filter through polystyrenedivinylbenzene column 

Instrument/Detector Liquid chromatograph Agilent 1100 with MS (API 4000 with ESI interface 

and MS detector) 

Column: 1) Synergi Fusion-RP80, 125x4.6 mm; 5 μm Phenomenex 

 2) Eclipse XDB-C8, 150x4.6 mm; 5 μm Agilent 

Mobile phase: 

A: water/ACN (9/1, v/v), containing 0.1 mL/L acetic acid. 

B: ACN, containing 0.1 mL/L acetic acid 

C: water/ACN (1/1, v/v), containing 0.1 mL/L acetic acid 

Ionization: ESI, positive 

MRM of transitions: 

BYI 08330-ketohydroxy-alcohol 334 to 298 m/z

BYI 08330-desmethyl-ketohydroxy 304 to 268 m/z 

BYI 08330-desmethyl-di-hydroxy 306 to 227 m/z. 

Standardization method Mixed stable isotope internal standards of BYI 08330-ketohydroxy-alcohol, 

BYI 08330-desmethyl-ketohydroxy and BYI 08330-desmethyl-di-hydroxy. 

Stability of std solutions All analytes are stable in standard solutions for at least 2 months of 

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

Retention times Column 1: BYI 08330-ketohydroxy-alcohol (~15.4 minutes), 

 BYI 08330-desmethyl-ketohydroxy (~16.8 minutes), 

 BYI 08330-desmethyl-di-hydroxy (~5.5 minutes). 

Column 2: BYI 08330-ketohydroxy-alcohol (~12.1 minutes), 

 BYI 08330-desmethyl-ketohydroxy (~14.3 minutes), 

 BYI 08330-desmethyl-di-hydroxy (~4.3 minutes). 
– 18 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.1-5. Characteristics for the Data-Gathering Analytical Method 00929 Used for the 

Quantitation of BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethylketohydroxy, and BYI 08330-desmethyl-di-hydroxy in Rotational Crop Plant 

Matrices.

Analyte(s) BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl-ketohydroxy, 

and BYI 08330-desmethyl-di-hydroxy 

Equipment ID Agilent 1100 HPLC system 

LOQ 0.02 ppm for each analyte in all plant matrices. 

LOD The LOD for all analytes was estimated to be at least 3-46% of the 

corresponding LOQ. 

Accuracy/Precision Range of percent recoveries ± coefficient of variation indicated 

acceptable accuracy/precision in the spiking levels of 0.02 ppm and 

0.20 ppm of each analyte for plant matrices. Mean recoveries of all 

analytes from all tested matrices were within the acceptable range of 

70-120% with RSD <11%. 

Reliability of the Method/ [ILV] An ILV was not conducted. 

Linearity The method/detector response was linear (r ≥0.997) within the range 

of 0.02 – 20 μg/L 

Specificity The control chromatograms generally have no peaks above the 

chromatographic background and the spiked sample 

chromatograms contain only the analyte peak of interest. Peaks 

were well defined and symmetrical. A second HPLC column was 

used for confirmation purposes. 

TABLE B.5.2.1-6. Extraction Efficiency of Method 00929 using Radiolabelled Samples of 

Swiss Chard and Wheat Straw from Confined Rotational Crop Study. 

Crops Swiss Chard (% extracted) Wheat Straw (% extracted) 

Method 

00929 

Confined crop 

rotation study 

Method 00929 Confined crop 

rotation study 

% TRRs extracted 89.2 91.3 90.5 93.7 

% of Compounds in the extract 

BYI 08330-desmethyl-di-hydroxy 6.8 7.7 22.2 25.5 

BYI 08330-ketohydroxy-alcohol 5.0 5.8 13.8 13.4 

BYI 08330-desmethyl-ketohydroxy 22.5 20.7 28.4 30.4

Enforcement Method 01084:

During the evaluation the residue definition for plant matrices has changed. The notifier submitted an

adequate analytical method which is evaluated by Austria for EU. 

Method 01084 is proposed as the enforcement method for the determination of residues of spirotetramat 

(BYI 08330), BYI08330-enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enolglucoside metabolites in/on plant material by HPLC-MS/MS. 

Principle of the Method:

Spirotetramat (BYI 08330) and its metabolites are extracted from potato (tuber), citrus (fruit), avocado (fruit), 

tomato (fruit) and hop (cone dried) using a mixture of acetonitrile/water. After filtration the extracts, aliquots 

will be diluted with a mixture of acetonitrile/water. 

The residues are quantified by reversed phase HPLC with electrospray and MS/MS-detection (external 

calibration using standard in matrix). 
– 19 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.1-7. Summary Parameters for the Analytical Enforcement Method 01084 Used for 

the Quantitation of Spirotetramat, BYI08330-enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy, and BYI08330-enol-glucoside metabolites in Plant 

Matrices.

Method ID Analytical method 01084 

Analyte(s) Spirotetramat, BYI08330-enol, BYI08330-ketohydroxy, BYI08330-monohydroxy, and BYI08330-enol-glucoside metabolites in/on plant material 

Extraction solvent/technique Acidic ACN/water mixture (4/1, v/v, containing 0.22 mL formic acid/L) 

Cleanup strategies Celite 545 and filtration 

Instrument/Detector Liquid chromatograph e.g. Agilent 1100 with Triple Quadrupole Tandem 

(MS/MS) e.g. API 4000 (AB MDS Sciex). 

Column: e.g.Zorbax, Eclipse XDB C8, 150 x 4.6 mm, 2.5μm, pre column 

YMC ProC18, VS 12nm, S-3μm, 10 x 2.1 mm id 

Mobile phase: 

Mobile Phase A: Water/acetonitrile (9/1, v/v) + 10 mmol/L 

ammonium formate + 220 μL formic acid/L 

Mobile Phase B: Acetonitrile + 10 mmol/L ammonium formate + 

220 μL formic acid/L 

Isocratic Pump: Acetonitrile/Water (1/1, v/v) + 10 mmol/L 

ammonium formate + 220 μL formic acid/L 

Different gradients are used for the determination of Spirotetramat, 

BYI08330-enol, BYI08330-ketohydroxy, and BYI08330-monohydroxy and the determination of BYI08330-enol-glucoside

Ionization: ESI, positive 

MRM of transitions (the second MRM was taken for confirmation): 

BYI08330 m/z 374 → 216 and m/z 374 → 302 

BYI08330-enol m/z 302 → 216 and m/z 302 → 270 

BYI08330-ketohydroxy m/z 318 → 268 and m/z 318 → 214 

BYI08330-monohydroxy m/z 304 → 254 and m/z 304 → 119 

BYI08330-enol-glucoside m/z 464 → 270 and m/z 464 → 216 

Standardization method Linear regression with 1/x weighting 

Stability of std solutions All analytes are stable in standard solutions up to 4 weeks of storage in a

refrigerator at 4°C ± 3°C under dark conditions. 

Retention times BYI 08330 approx. 4.7 min 

BYI08330-enol approx. 2.1 min 

BYI08330-ketohydroxy approx. 2.4 min 

BYI08330-mono-hydroxy approx. 2.0 min 

BYI08330-enol-glucoside approx. 1.7 min 

TABLE B.5.2.1-8. Characteristics for the Enforcement Analytical Method 01084 Used for the 

Quantitation of Spirotetramat Residues in Plant Matrices.

Analyte(s) Spirotetramat, BYI08330-enol, BYI08330-ketohydroxy, BYI08330-monohydroxy, and BYI08330-enol-glucoside metabolites in/on plant material

LOQ 0.01 mg/kg (expressed as parent equivalents) in avocado (fruit), orange 

(fruit), potato (tuber), and tomato (fruit) 

0.1 mg/kg in hop cones 

LOD 0.01 mg/kg in hop cones, 0.001 mg/kg in all other crops tested. 
– 20 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Accuracy/Precision Mean recoveries for each fortification level and analyte are between 70-

110 % for all matrices. 

Relative standard deviations were below 20 % for all analytes and sample 

materials. 

Detailed recovery information is reported below. 

Reliability of the Method/ [ILV] An ILV has not yet been performed on this method. 

Linearity The correlation between the injected amount of substance and the 

detector response was linear for solvent standards and matrix matched 

standards ranging from 0.025 to 10 μg/L. The correlation coefficients were 

between 0.9876 (lowest coefficient for BYl08330-enol-glucoside in solvent 

and was only for demonstration of matrix effects) and 0.9999 for all items. 

For quantitation of the recoveries only matrix matched standards were 

relevant. Representative linearity plots for matrix citrus are provided for 

each analyte (n = 5). 

Specificity Residues in control samples were below 30 % of the respective LOQ 

levels. The recoveries were not corrected for interferences. LC-MS/MS as 

a detection technique can be considered to be highly specific and 

therefore no confirmatory technique is required. 

Two MRM transitions were monitored for each analyte/matrix for 

quantitation and confirmation purposes. Chromatograms of control 

samples are available 

TABLE B.5.2.1-9. Validation data for analytical methods for the determination of residues of 

the active substance in food of plant origin. 

Analyte 

(each 

metabolite is 

expressed as 

parent 

equivalent)

Matrix 

Fortification 

Level 

[mg/kg] 

n

Mean 

Recovery

[%] 

RSD 

[%] 

Overall Mean 

Recovery 

[%] 

Overall RSD

[%] 

Hop 0.10 5 90 1.5 

Cone Dried 1.0 5 79 1.9 

85 7.0 

Avocado 0.01 5 94 2.6 

Fruit 0.10 5 93 3.8 

94 3.2 

Orange 0.01 5 93 2.6 

Fruit 0.10 5 91 0.6 

92 2.2 

Potato 0.01 5 93 1.3 

Tuber 0.10 5 89 1.8 

91 2.7 

0.01 5 92 1.5 

BYI 08330 

(1st MRM) 

Tomato 

Fruit 0.10 5 94 0.6 

93 1.4 

Hop 0.10 5 92 3.2 

Cone Dried 1.0 5 80 1.9 

86 8.1 

Avocado 0.01 5 93 2.5 

Fruit 0.10 5 94 2.3 

94 2.3 

Orange 0.01 5 90 2.9 

Fruit 0.10 5 91 1.4 

91 2.2 

Potato 0.01 5 92 2.1 

Tuber 0.10 5 87 1.1 

90 3.5 

BYI 08330 

(2nd MRM) 

Tomato 0.01 5 93 1.8 93 1.4 
– 21 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Analyte 

(each 

metabolite is 

expressed as 

parent 

equivalent)

Matrix 

Fortification 

Level 

[mg/kg] 

n

Mean 

Recovery

[%] 

RSD 

[%] 

Overall Mean 

Recovery 

[%] 

Overall RSD

[%] 

Fruit 0.10 5 93 1.0 

Hop 0.10 5 100 1.8 

Cone Dried 1.0 5 72 2.5 

86 17.2 

Avocado 0.01 5 93 3.2 

Fruit 0.10 5 88 1.0 

90 3.8 

Orange 0.01 5 87 1.4 

Fruit 0.10 5 89 1.2 

88 1.6 

Potato 0.01 5 88 1.2 

Tuber 0.10 5 90 0.9 

89 1.6 

0.01 5 89 1.0 

BYI08330- 

enol 

(1st MRM) 

Tomato 

Fruit 0.10 5 92 0.6 

91 1.9 

Hop 0.10 5 102 1.9 

Cone Dried 1.0 5 71 2.3 

87 18.9 

Avocado 0.01 5 95 1.5 

Fruit 0.10 5 88 1.0 

91 4.3 

Orange 0.01 5 86 2.1 

Fruit 0.10 5 88 1.3 

87 2.4 

Potato 0.01 5 88 1.9 

Tuber 0.10 5 90 0.8 

89 1.7 

0.01 5 89 1.8 

BYI08330- 

enol 

(2nd MRM) 

Tomato 

Fruit 0.10 5 91 0.6 

90 1.9 

Hop 0.10 5 97 5.0 

Cone Dried 1.0 5 72 2.1 

85 15.8 

Avocado 0.01 5 94 4.7 

Fruit 0.10 5 87 1.0 

91 5.2 

Orange 0.01 5 95 3.0 

Fruit 0.10 5 89 2.2 

92 4.1 

Potato 0.01 5 96 5.1 

Tuber 0.10 5 96 6.0 

96 5.3 

0.01 5 91 3.4 

BYI08330- 

ketohydroxy

(1st MRM 

Tomato 

Fruit 0.10 5 94 2.1 

92 3.1 

Hop 0.10 5 96 3.6 

Cone Dried 1.0 5 72 2.9 

84 15.9 

Avocado 0.01 5 91 2.5 

Fruit 0.10 5 91 2.1 

91 2.2 

Orange 0.01 5 92 2.2 

Fruit 0.10 5 91 1.7 

92 2.0 

Potato 0.01 5 94 4.2 

Tuber 0.10 5 99 6.9 

96 6.1 

0.01 5 96 4.2 

BYI08330- 

ketohydroxy

(2nd MRM 

Tomato 

Fruit 0.10 5 95 1.7 

96 3.1 

Hop 0.10 5 100 3.3 

Cone Dried 1.0 5 72 3.2 

86 17.2 

BYI08330- 

monohydroxy Avocado 0.01 5 97 2.1 92 5.3 
– 22 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Analyte 

(each 

metabolite is 

expressed as 

parent 

equivalent)

Matrix 

Fortification 

Level 

[mg/kg] 

n

Mean 

Recovery

[%] 

RSD 

[%] 

Overall Mean 

Recovery 

[%] 

Overall RSD

[%] 

Fruit 0.10 5 88 0.5 

Orange 0.01 5 87 3.6 

Fruit 0.10 5 90 1.0 

88 2.8 

Potato 0.01 5 93 2.0 

Tuber 0.10 5 94 1.7 

93 2.0 

0.01 5 93 1.7 

(1st MRM) 

Tomato 

Fruit 0.10 5 89 0.8 

91 2.6 

Hop 0.10 5 97 2.8 

Cone Dried 1.0 5 74 3.4 

85 14.4 

Avocado 0.01 5 98 3.6 

Fruit 0.10 5 91 1.0 

95 4.4 

Orange 0.01 5 87 3.5 

Fruit 0.10 5 90 1.0 

89 3.3 

Potato 0.01 5 89 4.9 

Tuber 0.10 5 104 1.5 

96 8.9 

0.01 5 95 2.1 

BYI08330- 

monohydroxy 

(2nd MRM) 

Tomato 

Fruit 0.10 5 91 2.0 

93 3.1 

Hop 0.10 5 106 6.2 

Cone Dried 1.0 5 85 4.8 

96 12.6 

Avocado 0.01 5 101 6.7 

Fruit 0.10 5 107 2.1 

104 5.6 

Orange 0.01 5 88 5.2 

Fruit 0.10 5 98 2.0 

93 7.1 

Potato 0.01 5 90 5.5 

Tuber 0.10 5 97 2.7 

93 5.4 

0.01 5 88 5.5 

BYI08330- 

Glucoside 

(1st MRM) 

Tomato 

Fruit 0.10 5 88 2.2 

88 4.0 

Hop 0.10 5 100 4.1 

Cone Dried 1.0 5 80 6.9 

90 13.0 

Avocado 0.01 5 101 6.8 

Fruit 0.10 5 103 3.1 

102 5.0 

Orange 0.01 5 84 4.4 

Fruit 0.10 5 94 4.5 

89 7.4 

Potato 0.01 5 94 3.7 

Tuber 0.10 5 99 5.5 

97 5.3 

0.01 5 85 3.0 

BYI08330- 

Glucoside 

(2nd MRM) 

Tomato 

Fruit 0.10 5 92 4.9 

89 5.6 

Conclusion:

The analytical method 01084 is sufficiently validated to monitor the active substance and the metabolites 

according to the current residue definition in plant material. 

The calibration range for hop should be up to 12 μg/L to fulfill the requirement that the upper calibration value 

should exceed at least 20% the highest measured level. The given value of10 μg/L can be accepted since

appropriate dilution can be carried out to be within the calibration range. 
– 23 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

An independent laboratory validation is not yet available. 

Multiresidue Method

 

Principle of the Method:

Residues of spirotetramat, BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy, BYI 08330-

enol-Glc, and BYI 08330-enol-GA were subjected to the appropriate steps of the multiresidue methodology 

described in the United States Food and Drug Administration (FDA) Pesticide Analytical Manual (PAM), 

Volume 1. The pertinent protocols tested are in TABLE B.5.2.1-9. 

TABLE B.5.2.1-10. Results of Multiresidue Methods Testing with Spirotetramat, BYI 08330-enol, 

BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy, BYI 08330-enol-Glc, and 

BYI 08330-enol-GA.

PAM 1 Protocols Results Comments 

A BYI 08330-mono-hydroxy was 

found to be naturally 

fluorescent. 

BYI 08330-mono-hydroxy is a minor metabolite and 

not being proposed as an enforcement target 

compound; therefore, no further testing was 

performed. 

B Not performed Test substances are not acids or phenols. 

C Ucceptable Nitrogen-specific modules DG5 and DG17 were 

unsuccessful at chromatographing the test 

substances due to no peak or the response was 

outside the relative retention time window. Nonspecific modules DG1, DG13 and DG18 were 

unsuccessful at chromatographing the test 

substances due to no peak, multiple peaks, or the 

response was outside the relative retention time 

window. 

D, E, F Not performed No further analyses were performed for Protocols D, 

E and F due to unsuccessful results with Protocol C. 

G Not performed Test substances are not substituted ureas. 

Conclusion:

Analytical method 00857 quantitating spirotetramat, BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-

mono-hydroxy and BYI 08330-enol-Glc was adequately validated on a wide variety of plant commodities and 

is suitable for data-gathering and enforcement purposes. Analytical method 00929 quantitating residues of 

BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl-ketohydroxy, and BYI 08330-desmethyl-di-hydroxy in 

rotational crops was adequately validated on wheat (grain, forage, straw and flour), cotton undelinted seeds, 

Swiss chard, peanut (oil and nutmeat), and sugar beets (roots and molasses). The extraction efficiencies of 

methods 00857 and 00929 were tested using samples containing aged radioactive residues from 

metabolism studies. The recoveries of the extracted radioactive residues of spirotetramat and the 

metabolites were in agreement with those generated in the metabolism studies. 

The test substances spirotetramat, and metabolites BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-

enol-Glc, and BYI 08330-enol-GA could not be determined by the FDA PAM Volume 1 multiresidue 

methods. 
– 24 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.2.2 Foodstuff of animal origin, feeding stuffs 

Report: KIIA 4.3/11, Freitag, Th., Wolters, A.; 2006 

Title: Analytical method 00966 for the determination of residues of BYI 08330, BYI08330-

enol, and BYI08330-enol-GA in/on matrices of animal origin by HPLC-MS/MS 

Report No & 

Edition No 

MR-150/05 

M-266199-01-2 

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

EU Guidance Document for generating and reporting methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/12, Duah, F.; 2006 

Title: Request for waiver of the requirement for radiovalidation of analytical method for the 

determination of BYI08330 residues in animal matrices 

Report No & 

Edition No 

RAFNX039 

M-277087-01-1 

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340, Residue 

Analytical Method-Extraction Efficiency 

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/13, Perez R; 2006 

Title: Independent method validation of Bayer method no. 00966 (report no. MR-150/05) 

BYI08330: Analytical method 00966 for determination of residues of BYI08330, 

BYI08330-enol and BYI08330-enol-glucuronide in/on matrices of animal origin by 

HPLC-MS/MS 

Report No & 

Edition No 

RAFNP009 

M-277298-01-1 

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

Report: KIIA 4.3/14, Coopersmith H; 2006 

Title: Analytical method for the determination of residues of BYI08330, BYI08330-enol 

and BYI08330-enol-GA in/on matrices of animal origin by HPLC-MS/MS 

(modification of analytical method 00966) 

Report No & 

Edition No 

RAFNP017 

M-277359-02-1 

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 

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

EU Guidance Document for generating and reporting methods of analysis 

SANCO/3029/99 

GLP Yes 

Report: KIIA 4.3/15 Freitag, Th., Wolters, A.; 2006 

Title: Analytical method 00969 for determination of residues of BYI08330-enol in/on 

matrices of animal origin by HPLC-MS/MS 

Report No & 

Edition No 

MR-160/05 

M-265407-01-1 

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

EU Guidance Document for generating and reporting methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

 PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 

 
– 25 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Report: KIIA 4.3/16 Bacher, R.; 2006 

Title: Independent laboratory validation of BCS Analytical Method 00969 for the 

determination of residues of BYI08330-enol in materials of animal origin 

Report No & 

Edition No 

P/B 964 G 

M-269571-01-1 

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

EU Guidance document for generating and reporting Methods of analysis 

SANCO/3029/99 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 

GLP Yes 
– 26 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Data-Gathering Method 00966

Principle of the Method:

Spirotetramat and the metabolites BYI 08330-enol and BYI 08330-enol-GA were extracted from the sample 

material (fat, liver, kidney) using a blender with an acidic acetonitrile/water mixture (7/3, v/v). Milk and 

muscle were extracted using a blender with acidic acetonitrile. After subsequent clean-up by exhausting the 

extract through a Varian MEGA BE-C18 cartridge, the corresponding stable isotopically labelled analytes 

were added as internal standards. Afterwards, the solution was made up to volume, diluted and subjected to 

HPLC-MS/MS. 

Bayer Method FN-006-A06-01

Analytical method 00966 was modified after an ILV. As recommended by the ILV laboratory, the method 

was modified (Bayer Method FN-006-A06-01) to provide an option to remove a cleanup step for livestock 

tissues; while milk, milk fat and whey samples are purified by C18 solid-phase extraction (SPE) prior to 

HPLC-MS/MS analysis. 

 

TABLE B.5.2.2-1. Summary Parameters for the Analytical Method Used for the Quantitation of 

Spirotetramat Residues in Livestock Matrices. 

Method ID Analytical Method 00966 Bayer Method FN-006-A06-01 

(modification of Analytical Method 00966) 

Analyte(s) Spirotetramat, BYI 08330-enol, BYI 08330–enol-GA 

Extraction 

solvent/technique 

Fat, liver & kidney - homogenize with acidic acetonitrile/water (7/3, v/v); Milk & 

muscle - homogenize with acidic acetonitrile. 

Cleanup strategies By filtering through a Varian MEGA BEC18 cartridge. After homogenization, livestock tissues 

were stirred well and allowed to separate 

until solids were at the bottom of the jar). 

Milk, milk fat or whey samples were 

cleaned up by filtering through a Varian 

MEGA BE-C18 cartridge. 
– 27 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.2-1. Summary Parameters for the Analytical Method Used for the Quantitation of 

Spirotetramat Residues in Livestock Matrices. 

Instrument/Detector Liquid chromatograph Agilent 1100 with 

mass spectrometer (API 4000 with ESI 

interface and mass spectrometric 

detector). 

Column: Agilent Zorbax EclipseXDB-C8, 5 μm, 150x4.6 mm i.d. 

with pre-column or equivalent. 

Mobile phase: 

Pump A: water/acetonitrile (9/1, 

v/v), containing 0.22 mL/L formic 

acid. 

Pump B: acetonitrile, containing 

0.22 mL/L formic acid. 

Pump C: water/acetonitrile 

 (1/1, v/v), containing 0.1 mL/L 

 acetic acid or formic acid. 

Ionization: ESI, positive 

1

st MRM Spirotetramat: 374 to 216 m/z

BYI 08330-enol: 302 to 216 m/z 

BYI 08330-enol-GA: 478 to 302 m/z 

2

nd MRM Spirotetramat: 374 to 302 m/z

BYI 08330-enol: 302 to 270 m/z 

BYI 08330-enol-GA: 478 to 216 m/z

Thermo Finnigan Surveyor instrument. 

Column: Gemini C6-Phenyl, 3 

μm, 50x2.0 mm i.d. with precartridge Serial Nos. 333946-3 & 

 347941-1. 

Mobile phase: 

A: water/acetonitrile (9/1, v/v), 

containing 0.22 mL/L formic acid. 

 B: acetonitrile, containing 0.22 

mL/L formic acid. 

Scanning mode: SelectedReaction Monitoring (SRM), 

negative ion mode. 

 Parent ion Daughter ion 

 Spirotetramat 374 to 216 

 BYI 08330-enol 302 to 216 

 BYI 08330-enol-GA 478 to 302 

Standardization 

method 

Mixed stable isotope internal standards of Spirotetramat, BYI 08330-enol and BYI 

08330-enol-GA 

Stability of std 

solutions 

Spirotetramat, BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy 

and BYI 08330-enol-GA are stable in standard solutions for at least four weeks of 

storage in a refrigerator at 4oC ± 3oC under dark conditions. 

Retention times Spirotetramat (~4.6 minutes), BYI 

08330-enol (~3.9 minutes), BYI 08330-

enol-GA (~1.9 minutes) 

Not Reported 
– 28 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.2-2. Characteristics for the Data-Gathering Analytical Method Used for the 

Quantitation of Spirotetramat Residues in Livestock Matrices.

Analyte(s) Spirotetramat, BYI 08330-enol, BYI 08330-enol-GA 

Equipment ID Agilent 1100 HPLC system 

LOQ 0.01 ppm for each analyte in all livestock matrices, except milk. 0.005 

ppm for each analyte in milk. 

LOD The LOD for all analytes was estimated to be 10 fold lower than the 

corresponding LOQ. 

Accuracy/Precision Range of percent recoveries ± coefficient of variation indicated acceptable 

accuracy/precision in the spiking levels of 0.01 ppm and 0.10 ppm for 

muscle (poultry & cattle), cattle kidney, liver & fat; and 0.005 ppm and 

0.050 ppm for milk. The percent recoveries from all matrices were 68-

101% for spirotetramat, 75-102% for BYI 08330-enol, and 69-105% for 

BYI 08330-enol-GA. Average recoveries ranged from 72% to 99% and 

relative standard deviations were <10%. 

Reliability of the Method/ [ILV] An ILV was conducted to verify the reliability of method No. 00966 for the 

determination of spirotetramat, BYI 08330-enol and BYI 08330-enol-GA 

residues in livestock matrices. The values obtained are indicative that 

method No. 00966 is reliable. 

Linearity The method/detector response was linear (coefficient of determination, r2= 

0.999) within the range of 0.02-20 ug/L. 

Specificity The control chromatograms generally have no peaks above the 

chromatographic background and the spiked sample chromatograms 

contain only the analyte peak of interest. Peaks were well defined and 

symmetrical. Two MRM transitions were monitored for each matrix and 

each analyte for quantitation and confirmation purposes. 

Independent Laboratory Validation

Analytical method 00966

An ILV was conducted by ADPEN Laboratories, Inc., Jacksonville, FL. Homogenized beef liver control 

samples were spiked with spirotetramat, BYI 08330-enol and BYI 08330-enol-GA at 0.01 ppm (LOQ), 0.02 

ppm and 0.10 ppm, and analyzed by HPLC-MS/MS according to method 00966. During the first trial, the 

method was followed as written. The recoveries of spirotetramat in beef liver were low at all spiking levels 

while the recoveries of BYI 08330-enol were high. The method was modified in that the extraction solvent 

was added within 5 minutes of spiking and each sample was extracted within 30 minutes. As well, the SPE 

cleanup step with the Varian MEGA BE-C18 was removed. The second ILV trial using the modified method 

was successful and supports the method LOQ of 0.01 ppm. At the second trial, individual recoveries of

spirotetramat, BYI 08330-enol, and BYI 08330-enol-GA from spiked beef liver ranged from 72% to 99%. 

Average recoveries ranged from 76% to 96% with RSD ≤5%. 
– 29 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.2-3. Recovery Results Obtained by an Independent Laboratory Validation of the 

Analytical Method 00966 for the Determination of spirotetramat Residues in 

 Livestock Matrices. 

Matrix Spiking Level 

(ppm) 

Recoveries Obtained 

(%) 

Mean Recovery ± SD (CV)

(%) 

Spirotetramat 

Beef liver 0.01 93, 85, 93, 91, 90 90 ± 3 (4) 

 0.02 93, 92, 93, 89, 96 93 ± 3 (3) 

 0.10 86, 90, 94, 96, 95 92 ± 4 (4) 

BYI 08330-enol 

0.01 99, 96, 94, 88, 88 93 ± 5 (5) 

Beef liver 0.02 91, 98, 99, 97, 93 96 ± 3 (3) 

0.10 91, 93, 97, 93, 98 94 ± 3 (3) 

BYI 08330-enol-GA 

0.01 80, 76, 79, 72, 76 76 ± 3 (4) 

Beef liver 0.02 76, 77, 77, 79, 76 77 ± 2 (1) 

0.10 72, 79, 76, 76, 82 77 ± 5 (4) 

Conclusion:

Analytical method 00966 (updated to FN-006-A06-01) quantitating spirotetramat, BYI 08330-enol and BYI 

08330-enol-GA was adequately validated and is suitable for data-gathering purposes in cattle and poultry 

tissues, milk and eggs. Although the proposed enforcement method 00969 measures only metabolite BYI 

08330-enol in livestock matrices, it was successfully validated in cattle milk, muscle and poultry eggs. A 

waiver was submitted by the applicant to remit the requirement for an extraction efficiency study. An 

extraction efficiency study was not considered necessary since method 00966 uses the same extraction 

solvents as those used in the livestock metabolism study. 
– 30 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Enforcement method 00969: 

Principle of the method:

BYI 08330-enol was extracted from egg and kidney samples using a blender with an acetonitrile/water 

mixture (7/3, v/v). Milk and muscle samples were extracted using a blender with acetonitrile. After 

subsequent clean-up of the extract through a Varian MEGA BE-C18 cartridge, the solution was made up to

volume, diluted and subjected to HPLC-MS/MS. 

TABLE B.5.2.2-4. Summary Parameters for the Analytical Enforcement Method Used for the 

Quantitation of BYI 08330-enol Residues in Livestock Matrices.

Method ID Analytical method 00969 

Analyte(s) BYI 08330-enol 

Extraction solvent/technique Kidney & egg - homogenize with acetonitrile/water (7/3, v/v) 

Milk & muscle - homogenize with acetonitrile 

Cleanup strategies By filtering through a Varian MEGA BE-C18 cartridge. 

Instrument/Detector same as described for method 00966 

Standardization method External standards; matrix-matched standards 

Stability of std solutions Spirotetramat-enol is stable in standard solutions for at least four weeks of 

storage in a refrigerator at 4oC ± 3oC under dark conditions. 

Retention times BYI 08330-enol (~3.8 minutes) 

TABLE B.5.2.2-5. Characteristics for the Enforcement Analytical Method 00969 Used for the 

Quantitation of BYI 08330-enol Residues in Livestock Matrices.

Analyte(s) BYI 08330-enol 

Equipment ID Agilent 1100 HPLC system 

LOQ 0.01 ppm (kidney, muscle, egg) 

0.005 ppm (milk) 

LOD 0.001 ppm (kidney, muscle, egg) 

0.0005 ppm (milk) 

Accuracy/Precision Range of percent recoveries ± coefficient of variation (≤6%) indicating 

acceptable accuracy/precision in the range of spiking levels of 0.01 ppm 

and 0.10 ppm for cattle kidney & muscle, and egg; 0.005 ppm and 0.05 

ppm for milk. 

Reliability of the Method/ [ILV] An ILV was conducted to verify the reliability of method No. 00969 for the 

determination of BYI 08330-enol residues in livestock matrices. The 

values obtained are indicative that method No. 00969 is reliable. The 

linearity range of ILV is 0.050 μg/L to 5.0 μg/L which is different from the 

range of 0.02 ng/mL to 20 μg/L reported for the primary method. 

Linearity The method/detector response was linear (r= 0.999) within the range of 

0.02-20 ug/L. 

Specificity The control chromatograms generally have no peaks above the 

chromatographic background and the spiked sample chromatograms 

contain only the analyte peak of interest. Peaks were well defined and 

symmetrical. 
– 31 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.2-6. Recovery Results from Method Validation of Livestock Matrices using the 

Enforcement Analytical Method 00969. Standards Were Prepared in 

Acetonitrile/Water (15/85, v/v containing 0.22 mL/L formic acid). 

Matrix Spiking Level 

(mg/kg) 

Recoveries Obtained Mean Recovery ± SD (CV)

BYI 08330-enol 

Cattle muscle 0.01 95, 98, 93, 92, 88 93 ± 4 (4) 

0.10 90, 84, 94, 95, 100 93 ± 6 (6) 

Cattle kidney 0.01 88, 95, 92, 91, 90 91 ± 3 (3) 

0.10 94, 95, 94, 95, 94 94 ± 1 (1) 

Cattle milk 0.005 99, 99, 96, 99, 103 99 ± 2 (3) 

0.050 97, 97, 94, 95, 97 96 ± 1 (1) 

Poultry egg 0.01 95, 88, 94, 91, 91 92 ± 3 (3) 

0.10 90, 94, 94, 94, 94 93 ± 2 (2) 

TABLE B.5.2.2-7. Characteristics for the Enforcement Analytical Method 00969 Used for the 

Quantitation of BYI 08330-enol Residues in Livestock Matrices.

Analyte(s) BYI 08330-enol 

Equipment ID Agilent 1100 HPLC system 

LOQ 0.01 ppm (kidney, muscle, egg) 

0.005 ppm (milk) 

LOD 0.001 ppm (kidney, muscle, egg) 

0.0005 ppm (milk) 

Accuracy/Precision Range of percent recoveries ± coefficient of variation (≤6%) indicating 

acceptable accuracy/precision in the range of spiking levels of 0.01 ppm 

and 0.10 ppm for cattle kidney & muscle, and egg; 0.005 ppm and 0.05 

ppm for milk. 

Reliability of the Method/ [ILV] An ILV was conducted to verify the reliability of method No. 00969 for the 

determination of BYI 08330-enol residues in livestock matrices. The 

values obtained are indicative that method No. 00969 is reliable. The 

linearity range of ILV is 0.050 μg/L to 5.0 μg/L which is different from the 

range of 0.02 ng/mL to 20 μg/L reported for the primary method. 

Linearity The method/detector response was linear (r= 0.999) within the range of 

0.02-20 ug/L. 

Specificity The control chromatograms generally have no peaks above the 

chromatographic background and the spiked sample chromatograms 

contain only the analyte peak of interest. Peaks were well defined and 

symmetrical. 

Independent Laboratory Validation

Analytical method 00969

An ILV for analytical method 00969 was conducted by PTRL Europe, Germany; using bovine meat, milk and 

poultry egg as the sample matrices. Control samples of meat and egg were spiked with BYI 08330-enol at 

0.01 and 0.10 ppm, and milk samples were spiked at 0.005 and 0.05 ppm. Due to significant modifications 

in the HPLC conditions, the first trial was considered unsuccessful. After consultation with the sponsor, the 

second trial was successful using HPLC gradient conditions similar to those given in the original method. 

The mean recoveries were 95-99% with RSD ≤8%. 
– 32 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.2.2-8. Recovery Results Obtained by an ILV of the Analytical Method 00969 for the 

Determination of BYI 08330-enol in Livestock Matrices

Matrix Spiking Level 

(mg/kg) 

Recoveries Obtained Mean Recovery SD (CV) 

BYI 08330-enol 

Cattle milk 0.005 93, 98, 94, 103, 103 98 ± 5 (5) 

0.050 99, 99, 99, 100, 93 98 ± 3 (3) 

Cattle muscle 0.01 106, 90, 88, 98, 100 97 ± 8 (8) 

0.10 96, 96, 96, 96, 101 97 ± 2 (2) 

Poultry egg 0.01 95, 91, 100, 96, 93 95 ± 4 (4) 

0.10 96, 100, 98, 102, 98 99 ± 2 (2) 

CONCLUSION 

Although the proposed enforcement method 00969 measures only metabolite BYI 08330-enol in livestock 

matrices, it was successfully validated in cattle milk, muscle and poultry eggs. 

Remark by EU: According to Section Residue only the metabolite BYI08330-enol is expected in animal 

matrices therefore the current residue definition for monitoring (i.e.BYI08330 and BYI08330-enol) should be 

changed. 
– 33 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.3 Analytical methods for the determination of residues in soil, water, sediment and air 

(Annex IIA 4.4 to 4.7, Annex IIIA 5.4 to 5.7) 

B.5.3.1 Soil (Annex IIA 4.4, Annex IIIA 5.4) 

Report: KIIA 4.4/01, Brumhard, B. 2006 (MR-06/034) 

Title: Analytical Method 00986 for the Determination of Residues of BYI08330 and 

BYI08330-enol in Soil by HPLC-MS/MS 

Report No & 

Edition No 

MR-06/034 (Method No. 00986) 

M-268586-01-1 

Guidelines: EC Guidance Document on Residue Analytical Methods, SANCO/825/00 rev.7 of 

March 17, 2004. BBA Guideline: Residue Analytical Methods for Post-Registration 

Control Purposes of July 21, 1998. Commission Directive 96/46/EC amending 

Council Directive 91/414/EEC of 16 July 1996. 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

GLP Fully GLP compliant - laboratory certified by German “Ministerium für Umwelt, 

Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen“. 

Testing 

Laboratory and 

Dates 

Bayer CropScience AG - Development - Residues, Operator and Consumer Safety, 

D-40789 Monheim, GER, conducted the work during the period of February to 

March 2006. Study completion date: 2006-03-22 

Report MR-06/034 provides a method description and validation data for the method 00986 that was 

developed for the determination of BYI08330 and its metabolite BYI08330-enol in soil. The objective of this 

work was to describe and to validate the residue analytical method 00986 by means of recovery tests in two 

soils spiked with each of the compounds listed above. The method is the recommended enforcement 

method for monitoring of BYI08330 residues in soil.

Principle of the method: 

Residues of BYI08330 and the metabolite BYI08330-enol are extracted from approx. 25 g of soil or sediment 

using an acidic extraction solution in the presence of cysteine hydrochloride and utilizing microwave 

extraction. The extraction solvent consists of a mixture of water (containing 8 g/L L-cysteine hydrochloride), 

acetonitrile, ethyl acetate and formic acid. An aliquot of the final extract is analyzed by LC-MS/MS. Possible 

matrix effects are eliminated by using matrix standard solution. Identification and quantification of the test 

items is done by high performance liquid chromatography using MS/MS detection in the Multiple Reaction

Monitoring (MRM) mode using instrumentation and conditions presented in the following table. 

HPLC System e.g. HP 1100 

HPLC Column e.g. Luna 3μ C8(2) 100A, size 50 mm x 2 mm, Part No. 00B4248-B0, Phenomenex, 

63741 Aschaffenburg, GER 

Injection Volume 20 μL 

Solvent A: Milli-Q water/acetic acid (1000/5, v/v) 

Solvent B: Acetonitrile 

Solvent Isocratic Pump 

for flushing the interface: Milli-Q water/acetonitrile/acetic acid (500/500/1, v/v/v) 

Oven Temperature: 40°C 

Flow rate (column): 0.2 mL/min 

Flow rate (into MS): 0.2 mL/min 

Gradient: 

Time [min] 0 2.9 3 5 7 8.1 11 11.1 13 

% A 85 85 70 55 55 35 35 85 85 

HPLC Method 

% B 15 15 30 45 45 65 65 15 15 

MS/MS System e.g. API 4000 with turbo-ionspray interface mass selective detector (MS/MS) (Perkin 

Elmer Sciex Instruments, Weiterstadt, GER) 
– 34 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

 BYI0833

0 

m/z 302 

BYI08330 

m/z 216 

BYI08330-enol 

m/z 270 

BYI08330-enol m/z 

216 

Q1 Mass [amu] 

Q3 Mass [amu] 

Dwell [msec] 

Ionisation Mode 

374.1 

301.8 

250 

TIS+ 

374.1 

215.9 

250 

TIS+ 

302.2 

269.7 

250 

TIS+ 

302.2 

215.8 

250 

TIS+ 

DP [V] 

CE [V] 

CXP [V] 

IS [V] 

EP [V] 

51 

25 

10 

5400 

10 

51 

47 

16 

5400 

10 

71 

29 

8 

5400 

10 

71 

37 

10 

5400 

10 

MS/MS 

operating 

parameters 

CAD 

CUR 

GAS 1 

GAS 2 

Turbo gas Temp. [°C] 

6 

10 

70 

70 

650 

6 

10 

70 

70 

650 

6 

10 

70 

70 

650 

6 

10 

70 

70 

650 

Retention time BYI08330 (m/z 302): approx. 9.9 min 

BYI08330 (m/z 216): approx. 9.9 min 

BYI08330-enol (m/z 270): approx. 7.2 min 

BYI08330-enol (m/z 216) : approx. 7.2 min 

IS: Ion Spray Voltage EP: Entrance Potential CXP: Collision Cell Exit Potential 

DP: Declustering Potential CE: Collision Energy 

TIS+: Turbo Ion Source in positive ion mode, i.e. production of positive ions 

Limit of quantification (LOQ) and Limit of Detection (LOD): 

The limit of quantification of the method is 5 μg/kg for BYI08330 and BYI08330-enol. The LOD was set to 1/3 

of the LOQ (LOD = 1.5 μg/kg). 

Calibration: 

Solvent standard solutions and matrix-mixed standard solutions containing BYI08330 and BYI08330-enol 

were measured in a concentration range of 0.5 to 50 μg/L corresponding to a concentration in soil of 2 to 200 

μg/kg. In this concentration range, the mass spectrometric detector showed linear correlation between 

concentration and peak area for BYI08330 and BYI08330-enol (a correlation coefficient of greater than 0.999 

for all tests). The slope values of the linearity curves show no matrix effects for BYI08330 m/z 302, BYI08330 

m/z 216, BYI08330-enol m/z 270 and BYI08330-enol m/z 216. 

Specificity: 

The blank values in all control samples were below 1.5 μg/kg (<1/3 x LOQ), demonstrating that no 

background level of BYI08330 and BYI08330-enol was present in the two test systems. 

Confirmatory method: 

Since HPLC-MS/MS and validation of two MRMs already offers maximum selectivity, i.e. specificity, a 

confirmatory procedure was considered to be unnecessary. 

Validation: 

The method was validated with a silt soil (Höfchen) and a sandy loam soil (Laacher Hof) by analyzing control 

samples and control samples spiked with BYI08330 and BYI08330-enol prior to the extraction procedure at 

the limit of quantification of 5μg/kg as well as 10 times this limit (50 μg/kg). The soils were spiked with a 

mixture of BYI08330 and BYI08330-enol. For method validation, 20 recovery experiments were conducted. 

Each sample was injected into the HPLC instrument. 
– 35 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Spiking levels as well as recoveries and relative standard deviations (RSD) are presented in tables B.5.3.1-1 

to B.5.3.1-4. Spiking levels are expressed as concentration of test item in wet soil. The listed data 

demonstrate the successful validation of the analytical method 00986 for the determination of BYI08330 and 

its metabolite BYI08330-enol in soil. 

TABLE B.5.3.1-1. Validation data for BYI08330 (m/z 302) in soils using the method 00986

Spiking 

[μg/kg] Soil Single Values [%] Mean [%] 

RSD 

[%] 

5 Höfchen 86 87 88 87 84 86 1.6 

5 Laacher Hof 93 92 95 94 92 93 1.4 

Mean of all 5 μg/kg single values 90 4.3 

50 Höfchen 98 96 100 97 99 98 1.8 

50 Laacher Hof 104 102 103 104 106 104 1.3 

Mean of all 50 μg/kg single values 101 3.3 

Mean of all Höfchen samples 92 6.8 

Mean of all Laacher Hof samples 98 5.7 

Overall mean 95 7.0 

TABLE B.5.3.1-2. Validation data for BYI08330 (m/z 216) in soils using the method 00986

Spiking 

[μg/kg] Soil Single Values [%] Mean [%] 

RSD 

[%] 

5 Höfchen 86 88 87 87 87 87 0.9 

5 Laacher Hof 91 90 95 92 92 92 2.1 

Mean of all 5 μg/kg single values 89 3.6 

50 Höfchen 96 94 97 96 94 95 1.5 

50 Laacher Hof 104 102 100 103 105 103 1.9 

Mean of all 50 μg/kg single values 99 4.2 

Mean of all Höfchen samples 91 5.2 

Mean of all Laacher Hof samples 97 6.1 

Overall mean 94 6.5 

TABLE B.5.3.1-3. Validation data for BYI08330-enol (m/z 270) in soils using the method 00986

Spiking 

[μg/kg] Soil Single Values [%] Mean [%] 

RSD 

[%] 

5 Höfchen 90 89 87 88 87 88 1.7 

5 Laacher Hof 99 95 98 97 90 96 3.7 

Mean of all 5 μg/kg single values 92 5.0 

50 Höfchen 94 93 96 94 94 94 1.1 

50 Laacher Hof 98 98 95 99 99 98 1.6 

Mean of all 50 μg/kg single values 96 2.4 

Mean of all Höfchen samples 91 3.6 

Mean of all Laacher Hof samples 97 2.9 

Overall mean 94 4.3 

RSD: relative standard deviation 

TABLE B.5.3.1-4. Validation data for BYI08330-enol (m/z 216) in soils using the method 00986

Spiking 

[μg/kg] Soil Single Values [%] Mean [%] 

RSD 

[%] 

5 Höfchen 92 91 89 90 88 90 1.8 
– 36 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

5 Laacher Hof 97 95 95 95 89 94 3.4 

Mean of all 5 μg/kg single values 92 3.6 

50 Höfchen 93 93 97 95 95 95 1.9 

50 Laacher Hof 96 96 94 97 100 97 2.4 

Mean of all 50 μg/kg single values 96 2.3 

Mean of all Höfchen samples 92 3.4 

Mean of all Laacher Hof samples 95 3.1 

Overall mean 94 3.6 

RSD: relative standard deviation 

Conclusion: 

The method 00986 (report MR-06/034) was developed for determination of BYI08330 and its metabolite 

BYI08330-enol in soil with an estimated limit of quantification of 5 μg/kg. This method was successfully 

validated according to the European requirements (96/46/EC of 16 July 1996 amending Directive 

91/414/EEC), the Guidance Document on Residue Analytical Methods (SANCO/825/00 rev.7 of March 17, 

2004) and BBA Guideline for Residue Analytical Methods of July 21, 1998: - the overall mean recovery of 

the method, which was determined in two soils at levels of 5 and 50 μg/kg each, was 95% for BYI08330 m/z 

302 (RSD: 7.0%), 94% for BYI08330 m/z 216 (RSD: 6.5%), 94% for BYI08330-enol m/z 270 (RSD: 4.3%) 

and 94% for BYI08330-enol m/z 216 (RSD: 3.6%). The repeatability expressed as the relative standard 

deviation (RSD) was lower than 7%. The method was found to be acceptable for monitoring of BYI08330 

residues in soil. 

Reference: In house laboratory validation of an analytical method for the determination 

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

BYI08330-MA-amide in soil and sediment by LC-MS/MS 

Author(s), year: Netzband, D. J.,2006 

Report/Doc. number: RAFNX012 

Guideline(s): US-EPA Test Guidelines OPPTS 850-7100 (1996), OPPTS 860.1340 (1996) 

GLP: Yes 

Deviations: None 

Validity: Study considered acceptable 

Material and methods:

Test substances: Spirotetramat, spirotetramat-enol, spirotetramat-ketohydroxy, spirotetramat-MAamide (all unlabelled) 

Internal standard: 13C3-Spirotetramat, 13C3-spirotetramat-enol, 13C3-spirotetramat-ketohydroxy, 13C3-

spirotetramat-MA-amide 

Test system: Two soil samples (North Rose, New York, study nr. MEFNY004, Lenz, 2006a, and 

Ephrata, Washington, study nr. MEFNY003, Lenz, 2006d) and one sediment 

(Columbia, SC); 

13 replicates for each soil, 7 replicates for the sediment 

Spiking: 5 and 25 μg kg-1 of each analyte separately 

Internal laboratory 

validation: 

An Intern laboratory validation (ILV) on the method has been performed with the 

soils ‘Höfchen’ and ‘Laacherhof’, spiking level not mentioned in the report 

Sample processing: Analytical method FN-002-S05-02 (updated to FN-002-S05-03): 20 g of soil are 

mixed with [25 mL of a solution of ethyl acetate (5 %, v/v) and cysteine 

hydrochloride (8 g L-1) in water, 0.5 mL formic acid, 25 mL acetonitrile], spiked 

with 1 ml of 13C internal standard solution and extracted by microwave extraction 
– 37 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

(70 °C, 30 min). Extraction (without addition of internal standard) is repeated once. 

Following extraction samples are filtered (0.45 μm) to await LC-MS/MS analysis. 

Analytical techniques: LC-MS/MS (screening for spirotetramat-enol dimer 1 and 2 possible) 

Limit of detection 

(LOD): 

Defined as: (standard deviation (spiking 5 μg kg-1) × t0.99) + average apparent 

residue in untreated control; 

where t0.99 is the one-tailed t-statistic at the 99 % confidence level of n-1 replicates 

(i.e. 2.68 in case of n = 13 and 3.14 in case of n = 7) 

Limit of quantification 

(LOQ): 

Defined as: (standard deviation (spiking 5 μg kg-1) × 5) + average apparent 

residue in untreated control 

Findings:

Calibration solutions of each analyte are stable over a minimum of 4 months when refrigerated. 

TABLE B.5.3.1-5. Mean recover and relative standard deviation (RSD) of analytes of freshly 

spiked soil and sediment samples. 

Soil Sediment 

Analyte 

Spiking 

level 

[μg kg-1] 

Overall mean 

[%] RSD [%] Overall mean [%] RSD [%] 

5 101 3.6 106 3.1 Spirotetramat 

25 106 1.6 108 3.2 

5 93 16.9 108 16.2 Spirotetramat-enol 

25 84 11.7 84 3.9 

Spirotetramat- 5 98 6.9 110 5.6 

ketohydroxy 25 103 5.7 105 7.2 

5 105 3.3 117 3.4 Spirotetramat-MA-amide

25 103 3.5 108 5.9 

TABLE B.5.3.1-6. Calculated LODs [μg kg-1] and LOQs [μg kg-1] for spirotetramat, 

spirotetramat-enol, spirotetramat-ketohydroxy and spirotetramat-MA-amide 

in soil and sediment samples. 

Matrix Analyte Calculated LOD 

[μg kg-1] 

Calculated LOQ 

 [μg kg-1] 

Spirotetramat 0.5 0.9 

Spirotetramat-enol 0.5 0.9 

Spirotetramat-ketohydroxy 2.1 3.9 

Soil 

Spirotetramat-MA-amide 0.9 1.7 

Spirotetramat 0.5 0.8 

Spirotetramat-enol 0.6 1.0 

Spirotetramat-ketohydroxy 2.7 4.4 Sediment 

Spirotetramat-MA-amide 1.0 1.5 

TABLE B.5.3.1-7. Results (mean recover and relative standard deviation, RSD) of the ILV 

using the soils ‘Höfchen’ and ‘Laacherhof’ freshly spiked with each analyte 

(spiking level not mentioned in the report). 

Höfchen Laacherhof 

Analyte Overall mean 

[%] RSD [%] Overall mean [%] RSD [%] 

Spirotetramat 94 1.8 92 1.4 

Spirotetramat-enol 91 2.8 91 1.7 
– 38 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Höfchen Laacherhof 

Analyte Overall mean 

[%] RSD [%] Overall mean [%] RSD [%] 

Spirotetramatketohydroxy 85 1.7 83 2.8 

Spirotetramat-MA-amide 89 13.7 94 13.1 

Conclusion:

The analytical method FN-002-S05-02 (updated to FN-002-S05-03 after revision) is suitable for the 

determination of spirotetramat and the metabolites spirotetramat-enol, spirotetramat-ketohydroxy and 

spirotetramat-MA-amide in soil and sediment by LC-MS/MS. The average recoveries for all analytes were 

between 70 and 120 % with a relative standard deviation (RSD) of less than 20 %. The method was shown 

capable of quantifying spirotetramat and its metabolites at the targeted LOQ of 5 μg kg-1

. 
– 39 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.3.2 Water (Annex IIA 4.5, Annex IIIA 5.5) 

Report: KIIA 4.5/01; Brumhard, B., 2006 (MR-131/03)

Title: Analytical Method 00836 for the Determination of BYI08330 and BYI08330-

enol in Drinking and Surface Water by HPLC-MS/MS and HPLC-UV 

Report No & 

Edition No 

MR-131/03 (Method No. 00836) 

M-250638-01-1 

Guidelines: EC Guidance Document on Residue Analytical Methods, SANCO/825/00 rev.7 of 

March 17, 2004. BBA Guideline: Residue Analytical Methods for Post-Registration 

Control Purposes of July 21, 1998. Commission Directive 96/46/EC amending 

Council Directive 91/414/EEC of 16 July 1996. 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

GLP Fully GLP compliant - laboratory certified by German “Ministerium für Umwelt, 

Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen“. 

Testing 

Laboratory and 

Dates 

Bayer CropScience AG - Development - Residues, Operator and Consumer Safety, 

D-40789 Monheim, GER, conducted the work during the period of February 2004 to 

April 2005. Study completion date: 2005-4-29 

Report MR-131/03 provides a method description and validation data for the method 00836 that was 

developed for the determination of BYI08330 and the metabolite BYI08330-enol in drinking and surface 

water. The objective of this work was to describe and to validate the residue analytical method 00836 by 

means of recovery tests in drinking and surface water spiked with each of the compounds listed above. The 

method is the recommended enforcement method for monitoring of BYI08330 residues in drinking and 

surface water. 

Principle of the method: 

Water samples are analysed by direct injection into an HPLC instrument without separate extraction and

clean-up steps. Identification and quantification of the test items is done by high performance liquid

chromatography using MS/MS detection in the Multiple Reaction Monitoring (MRM) mode or UV detection, 

using instrumentation and conditions presented in the following table. 
– 40 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

HPLC-MS/MS 

System 

e.g. HP 1100 with Quattro II with electrospray interface and mass selective detector 

(MS/MS) Waters GmbH, 65760 Eschborn, GER 

HPLC Column Synergi 4μ Fusion-RP 80, length 150 mm; i.d. 4.6 mm, article no. OOF-4424-EO; 

Phenomenex, 63768 Hosbach, GER 

Sample preparation HPLC-MS/MS 

Before analysis 20% of acetonitrile is added to the surface water samples. 

Additionally, acetic acid is added to a final concentration of 0.1 mL/L. These 

subsamples are directly injected into the HPLC instrument (HPLC-UV). 

Before analysis the water samples were adjusted to pH 3 with o-phosphoric acid. 

Additionally, 20% acetonitrile was added to the samples. These solutions were 

directly injected in HPLC instrument. 

Injection Volume 220 μL (if required it can be adapted to the concentration to be measured) 

Solvent A: Milli-Q water/acetonitrile/acetic acid (900/100/0.1, v/v/v) 

Solvent B: Acetonitrile/acetic acid (1000/0.1, v/v)

Oven Temperature: 40°C 

Flow rate (column): 1 mL/min 

Gradient: 

Time [min] 0 1 7 10 10.1 

% A 75 75 10 10 75 

HPLC Method 

% B 25 25 90 90 25 

MS/MS operating 

parameters 

Bath gas: 400 L/h nitrogen 5.0 

Nebulizer gas: 12 l/h nitrogen 5.0 

Reactant gas: Argon 5.0 (3.5 bar) 

Test item Detection 

time Principle Dwell[sec] 

Cone

[V] 

Collision 

energy 

[eV] 

Mass of 

parent ion

[amu] 

Mass of product 

ion 

[amu] 

BYI08330 5.0-9.0 ESP+ 0.5 42 32 374.23 215.95 

BYI08330-

enol 5.0-9.5 ESP+ 0.5 45 28 302.30 216.00 

 

Retention time BYI08330: approx. 8 min 

BYI08330-enol: approx. 6 min 

HPLC-UV System e.g. HP 1090 with HP1100 Series Wavelength Detector Agilent, 40880 Ratingen, 

GER 

HPLC Column Synergi 4μ Fusion-RP 80, length 150 mm; i.d. 4.6 mm, article no. OOF-4424-EO; 

Phenomenex, 63768 Hosbach, GER 

Injection Volume 250 μL (if required it can be adapted to the concentration to be measured) 

HPLC Method Solvent A: Milli-Q water adjusted to pH 3 with o-phosphoric acid 

Solvent B: Acetonitrile 

Oven Temperature: 40°C 

Flow rate (column): 1 mL/min 

Gradient: 

Time [min] 0 1 7 10 10.1 

% A 75 75 10 10 75 

% B 25 25 90 90 25 

UV operating 

parameter 

Wavelength: 210 nm 

Retention time BYI08330: approx. 8.5 min 

BYI08330-enol: approx. 7 min 

Limit of quantification (LOQ): 
– 41 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

The limit of quantification for BYI08330 and BYI08330-enol in surface water and drinking water is 0.05 μg/L, 

and the limit of detection is 0.02 μg/L, respectively. 

Calibration: 

In surface water the mass spectrometric detector showed linear response in the concentration range of 

approx. 0.04 μg/L to 12 μg/L for BYI08330 and BYI08330-enol with a correlation coefficient of > 0.9998. The 

UV detector showed linear response in surface water for both analytes in the concentration range of approx. 

5 μg/L to 1000 μg/L with a correlation coefficient of 1.0000. The MS/MS detection of BYI08330 and 

BYI08330-enol is affected by the matrix. The peak area of BYI08330 and BYI08330-enol in a surface water 

sample containing 0.5 μg/L was reduced to approx. 73% and 69%, respectively, of the corresponding peak

area in Milli-Q-water. The UV detection of BYI08330 and BYI08330-enol is not affected by the matrix. 

Specificity: 

The blank values of all control samples were below 0.3 x LOQ. 

Repeatability: 

Repeatability testing of MS/MS detection of BYI08330 and BYI08330-enol in drinking and surface water 

samples spiked at concentrations of 0.05 μg/L and 0.5 μg/L yielded a relative standard deviation for the peak 

area in the range of 1.4% and 6.5%, respectively. The relative standard deviation for the retention time was < 

0.2% for both analytes at both spiking levels. 

Repeatability testing of UV detection of BYI08330 and BYI08330-enol in surface water samples spiked at

concentrations of approx. 5 μg/L and 1000 μg/L yielded a relative standard deviation for the peak area in the 

range of 0.4% and 3.1%. The relative standard deviation for the retention time was < 0.1 % in each case. 

Confirmatory method: 

Since HPLC-MS/MS already offers maximum selectivity, i.e. specificity, a confirmatory procedure was 

considered to be unnecessary. 

Validation using MS/MS-detection: 

For method validation surface water from the river Rhine sampled in Leverkusen-Hitdorf and drinking water 

sampled in Monheim am Rhein, Bayer CropScience AG, building 6610, was used. For method validation 

surface and drinking water samples were spiked with BYI08330 and BYI08330-enol at 0.05 μg/L and at 0.5

μg/L. These test solutions were injected ten times each into the HPLC-MS/MS instrument. The peak areas

for BYI08330 and BYI08330-enol were determined and are listed in table B.5.3.2-1 and B.5.3.2-2. The 

relative standard deviation for the peak area of BYI08330 in surface water was 3.4% (0.05 μg/L) and 1.4% 

(0.5 μg/L), and in drinking water 6.5% and 5.4%, respectively. The relative standard deviation for the

retention time was always < 0.1%. The relative standard deviation for the peak area of BYI08330-enol in 

surface water was 2.7% (0.05 μg/L) and 1.7% (0.5 μg/L), and in drinking water 6.1% and 2.6%, respectively. 

The relative standard deviation for the retention time was < 0.2%, always 

TABLE B.5.3.2-1. Validation data for BYI08330 and BYI08330-enol in surface water using the 

method 00836 (MS/MS-detection) 

Peak area Retention time 

Spiking level (μg/L) Individual values 

(area counts) 

Mean 

(area counts) 

RSD 

(%) 

Mean 

(min) RSD (%)

BYI08330 

0.05 

88 / 89 / 84 / 88 / 81 

81 / 84 / 84 / 83 / 83 85 3.4 7.6 <0.1 

BYI08330 

0.5 

809/824/811 / 794/821 

806 / 797 / 799 / 790 / 798 805 1.4 7.6 <0.1 

BYI08330-enol 83 / 80 / 81 / 79 / 82 81 2.7 5.9 0.2
– 42 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Peak area Retention time 

Spiking level (μg/L) Individual values 

(area counts) 

Mean 

(area counts) 

RSD 

(%) 

Mean 

(min) RSD (%)

0.05 85 / 84 / 82 / 79 / 79 

BYI08330-enol 

0.5 

847/821 / 846/859/839 

842/855/831 / 81 5/851 841 1.7 5.9 0.1 

TABLE B.5.3.2-2. Validation data for BYI08330 and BYI08330-enol in drinking water using the 

method 00836 (MS/MS-detection) 

Peak area Retention time 

Spiking level (μg/L) Individual values 

(area counts) 

Mean 

(area counts) 

RSD 

(%) 

Mean 

(min) RSD (%)

BYI08330 

0.05 

38 / 35 / 38 / 38 / 41 

36 / 34 / 37 / 33 / 35 37 6.5 8.3 0.1 

BYI08330 

0.5 

333/329/323/322/312 

31 1 / 302 / 297 / 293 / 

282 

310 5.4 8.3 <0.1 

BYI08330-enol 

0.05 

38 / 35 / 39 / 35 / 32 

38 / 37 / 36 / 35 / 39 36 6.1 6.7 0.1 

BYI08330-enol 

0.5 

340 / 329 / 343 / 337 / 326

324/323/322/323/319 329 2.6 6.7 0.1 

Because of the direct measurement of spiked samples without separate extraction and clean-up steps, it is 

not possible to determine recovery rates and an estimate of the accuracy of the analytical technique was 

made by an assessment of the linearity of matrix calibration and by determination of the reproducibility of 

sample analysis. However, for additional demonstration of the reliability of the method, the validation 

samples were evaluated like recovery rates. From the single peak area of each sample listed, the 

percentages in Table 4.5-1 to Table 4.5-4 were calculated relative to the corresponding mean area, which 

was set at 100%. 

TABLE B.5.3.2-3. Percentages of BYI08330 and BYI08330-enol found in the spiked surface water 

samples using method 00836 (MS/MS-detection) 

Percentage found 

Spiking level (μg/L) Individual values 

(%) 

Mean 

[%) 

RSD 

(%) 

BYI08330 

0.05 

104 / 105 / 99 / 104 / 96 

96 / 99 / 99 / 98 / 98 100 3.4 

BYI08330 

0.5 

101 / 102 / 101 / 99 / 102 

100 / 99 / 99 / 98 / 99 100 1.4 

BYI08330-enol 

0.05 

102 / 98 / 100 / 97 / 101 

104 / 103 / 101 / 97 / 97 100 2.7 

BYI08330-enol 

0.5 

101 / 98 / 101 / 102 / 100 

100 / 102 / 99 / 97 / 101 100 1.7 

The MS/MS detection of BYI08330 and BYI08330-enol is affected by the matrix. The peak area of BYI08330

in a surface water sample containing 0.5 μg/L was reduced to approx. 73% of the corresponding peak area 

in Milli-Q-water and the peak area of BYI08330-enol in a surface water sample (0.5 μg/L) was reduced to 

approx. 69% of corresponding peak area in Milli-Q-water. The matrix effect was determined by four injections 

of each solution. 
– 43 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE B.5.3.2-4. Percentages of BYI08330 and BYI08330-enol found in the spiked drinking 

water samples using method 00836 (MS/MS-detection) 

Percentage found 

Spiking level (μg/L) Individual values 

(%) 

Mean 

[%) 

RSD 

(%) 

BYI08330 

0.05 

104 / 96 / 104 / 104 / 112 

99 / 93 / 101 / 90 / 96 100 6.5 

BYI08330 

0.5 

107 / 106 / 104 / 104 / 101

100 / 97 / 96/ 94 / 91 100 5.4 

BYI08330-enol 

0.05 

104 / 96 / 107 / 96 / 88 

104 / 102 / 99 / 96 / 107 100 6.1 

BYI08330-enol 

0.5 

103 / 100 / 104 / 103 / 99 

99 / 98 / 98 / 98 / 97 100 2.6 

Validation using UV-detection: 

For method validation surface water samples of river Rhine were spiked with BYI08330 and BYI08330-enol

at about 5 and 1000 μg/L. These test solutions were injected ten times each into the HPLC-UV instrument. 

The peak areas and retention times for BYI08330 and BYI08330-enol were determined and are listed in 

Table 16 and Table 17 of report MR-131/03. The relative standard deviation (RSD) for the peak area of 

BYI08330 in surface water was 1.6% (5 μg/L) and 0.4% (1000 μg/L), respectively, and the RSD for the 

retention time was < 0.1 % for both concentrations. The relative standard deviation (RSD) for the peak area 

of BYI08330-enol in surface water was 3.1% (5 μg/L) and 0.6% (1000 μg/L), respectively, and the RSD for 

the retention time was < 0.1 % for both concentrations. Because of the direct measurement of spiked 

samples without separate extraction and clean-up steps it is not possible to determine recovery rates and an 

estimate of the accuracy of the analytical technique was made by an assessment of the linearity of matrix 

calibration and by determination of the reproducibility of sample analysis. However, for additional 

demonstration of the reliability of the method, the validation samples were evaluated like recovery rates. 

From the single peak area of each sample the percentages listed in table B.5.3.2-5 were calculated relative 

to the corresponding mean area, which was set as 100%. 

TABLE B.5.3.2-5. Percentages of BYI08330 and BYI08330-enol found in the spiked surface water 

samples using method 00836 (UV-detection) 

Percentage found 

Spiking level 

(μg/L) 

Individual values 

(%) 

Mean 

[%) 

RSD 

(%) 

BYI08330 

5 

99 / 99 / 99 / 98 / 102 

101 / 102 / 101 / 99 / 99 100 1.6 

BYI08330 

1000 

101 / 100 / 100 / 100 / 99 

99 / 100 / 100 / 100 100 0.4 

BYI08330-enol 

5 

97 / 100 / 98 / 100 / 100 

107 / 101 / 103 / 96 / 98 100 3.1 

BYI08330-enol 

1000 

100 / 101 / 100 / 100 / 99 

99 / 100 / 100 / 100 / 100 100 0.6 

Storage stability: 

For examination of the storage stability surface water samples spiked separately with about 0.5 μg 

BYI08330/L and BYI08330-enol/L each were stored in a freezer at < -18 °C and in a refrigerator at < 6 °C for 

a period of about 4 months. Taking the pH-dependent hydrolytic stability of the two analytes into account the 

water samples spiked with BYI08330 were adjusted to pH 3 and the water samples containing BYI08330-

enol were kept at its natural pH (ca. pH 8). Prior to storage, all solutions were diluted with 20% of acetonitrile 
– 44 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

as outlined in earlier. A set of three samples was analyzed after 140 days of storage. The results showed 

that under freezer and refrigerator conditions BYI08330 and BYI08330-enol were stable for a storage period 

of at least 140 days. The mean recoveries were in the range of 92 to 107% for BYI08330 and in the range of 

76 to 113% for BYI08330-enol. 

Conclusion: 

The method 00836 (report MR-131/03) was developed for determination of BYI08330 and its metabolite 

BYI08330-enol in surface and drinking water with an estimated limit of quantification of 0.05 μg/L. This 

method was successfully validated according to the European requirements (96/46/EC of 16 July 1996 

amending Directive 91/414/EEC), the Guidance Document on Residue Analytical Methods (SANCO/825/00 

rev.7 of March 17, 2004) and BBA Guideline for Residue Analytical Methods of July 21, 1998: 

The method was assessed to be acceptable for monitoring of BYI08330 and BYI08330-enol residues in 

surface and drinking water. 

However, some points need clarification: 

Method preparation (surface water):

The notifier is asked to clarify: Were the samples spiked with active ingredient before or after filtration of the 

dry residue 

LC-MS/MS-detection: 

The calibration/linearity data for drinking water should be submitted. 

The described matrix effect for reducing the values to 73% and 69% respectively must be clarified in detail. 

The notifier is requested to submit raw data. 

UV-detection: 

The calibration range should extend over a range to the lowest and highest nominal concentration at least ± 

20%. For surface water the lowest measured concentration for calibration is 5.292 μg/L for BYI 8330 and 

5.133 μg/L for BYI 8330-enol respectively. Therefore the LOQ of 5.0 μg/L is not covered by the calibration 

range according to the EU guideline SANCO 825/00. This is of concern since all ecotoxicological studies 

have been investigated with the LC-UV method. 

B.5.3.3 Sediment (Annex IIA 4.6, Annex IIIA 5.6) 

Refer to document MR-06/034 from point 4.4 (KIIA 4.4/01). 

B.5.3.4 Air (Annex IIA 4.7, Annex IIIA 5.7) 

Not required or reviewed by the Canada PMRA. 

Required according to Directive 91/414/EEC and evaluated by Austria (AGES) for EU. 

Report: KIIA 4.7/01; Stupp, H.-P.; 2006 (MEF-06/119) 

Title: Method for the Determination of BYI08330 in Air by HPLC-UV and 

Confirmation by DAD (UV Spectra Match) 

Report No & 

Edition No 

MEF-06/119 (Method No. 01015) 

M-274784-01-1 

Guidelines: EU Directive 91/414/EEC Annex II (Part A, Section 4.2), as amended by 

Commission 

Directive 96/46/EC. EC Guidance document on residue analytical methods, 

SANCO/825/00 rev. 7 17/03/04. 

US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 

GLP Fully GLP compliant - laboratory certified by German “Ministerium für Umwelt, 

Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen“. 

Testing Bayer CropScience AG, Metabolism and Environmental Fate, D-40789 Monheim, 
– 45 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Laboratory and 

Dates 

GER, 

conducted the study during the period of February to March 2006. Study completion 

date: 

2006-06-06 

Validity Acceptable for the determination of spirotetramat (BYI08330) 

The objective of this study was to describe and to validate the residue analytical method by means of 

recovery tests in ambient and with warm, humid air (approx. 35°C, 80% relative humidity) spiked with 

BYI08330. In addition, a test with the major metabolite BYI08330-enol was included and reported. 

Test material:

Air at elevated (35 °C, 80% relative humidity) climatic conditions. 

Spiked analyte:

Spirotetramat 

Analyte(s) determined as:

Spirotetramat 

Principle of the method:

The air to be analysed is passed through commercial sealed cartridges at a flow rate of about 2 L/min for 6 

hours (total volume of 720 L). The sampling tubes comprise two portions of porous adsorbent polymer 

(Tenax®) separated with glass wool: the front portion (portion A, 100 mg adsorption material) is intended for 

collection of the analyte and the back portion (portion B, 50 mg adsorption material) allows control of 

breakthrough. Following sampling, the tubes are directly processed or are closed with plastic caps and are 

kept cool. The glass wool separating both layers and the upper glass wool facing the inlet of the tube are 

processed together with the front portion. The individual layers are removed from the glass tube and passed 

into separate 10-mL vials with snap cover. The glass tube is rinsed with 4 mL of acetonitrile. The front 

portion is mixed with these 4 mL of solvent and the back portion is mixed with 4 mL of acetonitrile, also. The 

test item is extracted from the adsorption material by shaking a laboratory shaker (about 300 rpm) for 30 

minutes. For HPLC analysis (i.e. using UV detection with external standard) of the upper LOQ samples the 

extracts were diluted 1:100 (v/v) with acetonitrile prior to injection in order to be in the same concentration 

range for analysis quantified by liquid chromatography. Instrumentation and conditions are presented in the 

following table. 
– 46 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

HPLC System e.g. Agilent 1100 with DA-detector and stationary mixer (vol. ca 1 mL) 

Detection wavelength at 254 nm (BW 8 nm), reference 550 nm (BW 80 nm). 

HPLC Column e.g. Purosphere Star 18e Fa. Merck 531455; length = 250 mm, inner diameter = 

4.6 mm, particle size =5 μm. Pre-column: Purosphere RP 18 (Lichrocart 4-4, Fa. 

Merck) 

Injection Volume 100 μL (samples were injected in duplicate) 

HPLC Method Solvent A: 0.01% acetic acid in water 

Solvent B: Acetonitrile 

Oven Temperature: 25°C 

Flow rate (column): 1.0 mL/min 

Linear gradient: 

Time (min) %A %B 

0.0 100 0 

5.0 100 0 

45.0 40 60 

55.0 0 100 

60.0 0 100 

65.0 100 0 

70.0 100 0 (post time 10 min) 

Retention time of BYI08330: 49.1 min 

Findings:

Specificity: Interferences in blank samples of max. 0.41 μg/m3

 [i.e. 4.1% of 10 μg/m3 (LOQ)] were found 

which is acceptable. No confirmatory technique is necessary since spirotetramat is successfully validated in 

soil and water matrices. 

Calibration/Linearity: The detector response was linear from 0.54 μg/mL to 5.4 μg/mL (5 levels, r2 = 0.9987). 

Samples of high spiking concentration were diluted 1:100 prior to injection. 

LOQ: 10 μg/m3 air. 

Accuracy: Mean recoveries are between 70-110 % at each spiking level with a RSD < 20 %. 

Breakthrough behaviour: No breakthrough was observed at both spikings (10 and 1000 μg/m3

). 

Validation data are summarised in table B.5.3.4-1. 

Spiked analyte:

Spirotetramat-enol 

Stability of spirotetramat-enol (BYI08330-enol) adsorbed onto 5 different materials (Tenax, XAD-2, Charcoal, 

Florisil and Silica Gel) was tested under same conditions as described for spirotetramat (BYI08330). 

Desorption recoveries at a spiking of 10 μg/m3 are either too low (20 and 33.1% mean recoveries) or non 

detectable due to complete degradation. 
– 47 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

TABLE 5.3.4-1: Validation data for spirotetramat (BYI08330) residues in air 

References Determined 

analyte 

Detection 

method 

Matrix LOQ

[μg/m

3

] 

Spiking 

level 

[μg/m3] 

Mean 

recovery 

[%] 

RSD 

 [%] 

n 

elevated temperature and humidity (35°C and 80% RH)

10 105.7 3.2 5 

Stupp, H.-

P.; 2006 

(MEF06/119) 

BYI08330 HPLC-UV 

Air 10 

1000 105.4 1.22 5 

Conclusion:

The method is successfully validated for the determination of spirotetramat (BYI08330) in air. According to 

Section Toxicology an AOEL systemic of 0.05 mg/kg bw/d was set which results in a value of 0.015 mg/m3

air (equivalent to 15 μg/m3

) for the concentration C with respect to the calculation of guidance document 

825/00. This concentration is covered by the LOQ set at 10 μg/m3. 

Adsorption tubes containing about 20 μg/m3 BYI08330 (i.e. 2 times LOQ) were stable in a refrigerator at 

about +2/+6 °C and in a deep-freezer at about -21/-24 °C for 8 days after sampling. 

The method is not suitable for determination of the main degradation product BYI08330-enol which is not 

included in the current residue definition in air. 

B.5.4 Analytical methods for the determination or residues in body fluids and tissues 

(Annex IIA 4,8; Annex IIIA 5.8) 

Not applicable, as compounds are not classified as toxic (T, T+). 
– 48 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.5 Evaluation and assessment 

The analytical methods provided for the analysis of the technical material and environmental samples have 

been assessed to be fully validated and acceptable. 

B.5.5.1 Analytical methods for technical active substance and formulation analysis 

Analytical methodology is available for the determination of the active substance and the impurities in the 

technical material. 

All methods are fully validated. No CIPAC method exists at the time of evaluation. 

Table 5.5.1-1: Summary of analytical methods for technical active substance and 

formulation analysis 

Matrix Analyte Type of method Validation References

Technical active 

substance 

Spirotetramat HPLC-UV 

Method 

AM001104MP1 

Full Hake, 2004 

HPLC-UV 

AM001204MP2 

Full Hake, 2006 

GLC (Headspace)

Method 

AM003406MP1 

Full Kraemer, 2006 

Technical active 

substance 

Impurities 

Karl Fischer 

titration 

Internationally 

accepted method 

CIPAC MT 30.5 

Technical active 

substance 

Formulation HPLC-UV 

Method 

AM002104MF1 

Full Odenahl 2005 

B.5.5.2 Analytical methods for residues 

The submitted methods enable the enforcement of the following relevant residue definitions and limits (at the 

time of evaluation). 

Table 5.5.2-1: Overview of accepted residue analytical methods 

Matrix type/ 

crop group 

Primary Method Analyte Confirmatory 

method 

Independent Lab 

Validation 

(if appropriate) 

Hope (cone dried) 

Avocado (fruit) 

Orange (fruit) 

Potato (tuber) 

Tomato (fruit) 

Schöning R., 

Willmes J. (2008) 

LC-MS/MS 

BYI08330 

BYI08330-enol 

BYI08330-

ketohydroxy 

BYI08330-

monohydroxy 

BYI08330-enolglucoside 

Not necessary Not yet available 

Animal origin Freitag, Th., BYI08330-enol Not necessary Bacher R; 2006 
– 49 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Matrix type/ 

crop group 

Primary Method Analyte Confirmatory 

method 

Independent Lab 

Validation 

(if appropriate) 

Wolters, A.; 2006 

LC-MS/MS 

Soil Brumhard B.; 2006

LC-MS/MS 

Spirotetramat Not necessary – 

Drinking water Spirotetramat Not necessary – 

Surface water 

Brumhard B.; 2006

LC-MS/MS Spirotetramat Not necessary – 

Air Stupp H.-P.; 2006 

LC-UV 

Spirotetramat Stupp H.-P.; 

2006 

LC-DAD 

(not 

necessary) 

– 

Table 5.5.2-2: Summary of analytical methods covering relevant residue definitions and 

limits 

Matrix Residue definition LOQ Residue limit 

Avocado (fruit) 

Orange (fruit) 

Potato (tuber) 

Tomato (fruit) 

0.01 mg/kg 

for each 

analyte 

Hope (cone dried) 

BYI08330 and the 4 

metabolites BYI08330-enol, 

BYI08330-ketohydroxy, 

BYI08330-monohydroxy, and 

BYI08330 enol-glucoside, all 

metabolites expressed as 

BYI08330. 

0.1 mg/kg 

for each 

analyte 

Proposed MRLs in mg/kg: 

Tomatoes 2.0 

Peppers 2.0 

Aubergines (egg plants) 2.0 

Cucumbers 0.2 

Courgettes 0.2 

Melons 0.2 

Pumpkins 0.2 

Watermelons 0.2 

Flowering brassica 1.0 

Brussels sprouts 0.3 

Head cabbage 0.5 

Kale 2.0 

Kohlrabi 2.0 

Lettuce 5.0 

Hops 10.0 

Milk 0.005 mg/kg 

BYI08330-enol 

calculated and 

expressed as 

BYI08330 

parent 

equivalents 

Proposed MRLs in mg/kg: 

Milk

Meat and egg 

BYI08330 and BYI08330-enol 

0.01 mg/kg 

BYI08330-enol 

calculated and 

expressed as 

BYI08330 

parent 

Proposed MRLs in mg/kg: 

Liver, meat

Meat by products 

(except liver)
– 50 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Matrix Residue definition LOQ Residue limit 

equivalents 

Soil Spirotetramat 5 μg/kg LC50 earthworm >1 g/kg soil dry 

weight 

NOEC is not required by section 

ecotoxicology. 

Drinking water Spirotetramat 0.05 μg/L 0.1 μg/L EU drinking water limit 

Surface water Spirotetramat 0.05 μg/L NOEC Chironomus riparius 

< 0.53 mg/L 

LC50 Chironomus riparius 

1.30 mg/L 

Air Spirotetramat 10 μg/m3 15 μg/m3 based on a proposed 

AOELsystemic of 0.05 mg/kg 

bw/day 

Body fluids Not necessary, the active substance is not classified as toxic or highly toxic 
– 51 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

B.5.6 References relied on 

Annex 

point/ 

reference 

number 

Author(s) Year 

Title 

Source (where different from 

company) 

Company, Report No 

GLP or GEP status (where relevant)

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

Annex II Data and Information 

KIIA 4.2.1 

/01 

Hake, G. 2004 Spirotetramat (BYI08330) - Assay of 

Technical Grade Active Ingredient - 

HPLC - External Standard 

Bayer CropScience AG, 

Report No.: AM001104MP1, 

Edition Number: M-084385-01-1 

Date: 07.07.2004 

Non GLP, unpublished 

Yes BCS 

KIIA 4.2.1 

/02 

Hake, G. 2004 Validation of HPLC-method 

AM001104MP1 - BYI08330 Assay of 

technical grade active ingredient 

Bayer CropScience AG, 

Report No.: VB2-AM001104MP1, 

Edition Number: M-084403-01-1 

Date: 07.07.2004, Amended: 

09.11.2006 

Non GLP, unpublished 

Yes BCS 

KIIA 4.3 /18 Schöning, R., 

Willems, J., 

2008 Analytical method 01084 for the 

determination of residues of 

spirotetramat (BYI 08330), BYI08330-

enol, BYI08330-ketohydroxy, 

BYI08330-mono-hydroxy and 

BYI08330-enol-glucoside metabolites 

in/on plant material by HPLC-MS/MS 

Bayer CropScience AG, 

Report No.: MR-08/007, 

Edition Number: M-298287-01-1 

Method Report No.: MR-08/007 

Date: 17.08.2005, Amended: 

02.06.2006 

GLP, unpublished 

Yes BCS 
– 52 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Annex 

point/ 

reference 

number 

Author(s) Year 

Title 

Source (where different from 

company) 

Company, Report No 

GLP or GEP status (where relevant)

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

KIIA 4.3 /08 KLimmek, S. 2005 BYI 08330-enol: Examination of the 

applicability of DFG merhod S 19 

(extended and revised revision) for the 

determination of residues of BYI 

08330-enol 

Specht & Partner Chemische 

Laboratorien GmbH, Hamburg, 

Germany 

Bayer CropScience AG, 

Report No.: BAY-0519, 

Edition Number: M-257512-01-1 

Method Report No.: BAY-0519 

Method Report No.: G05-0104 

Date: 08.09.2005 

GLP, unpublished 

Yes BCS 

KIIA 4.3 /15 Freitag, T.; 

Wolters, A. 

2006 Analytical method 00969 for the 

determination of residues of BYI08330-

enol in/on matrices of animal origin by 

HPLC-MS/MS 

Bayer CropScience AG, 

Report No.: 00969, 

Edition Number: M-265407-01-2 

Method Report No.: MR-160/05 

Date: 18.01.2006 

GLP, unpublished 

Yes BCS 

KIIA 4.3 /16 Bacher, R. 2006 Independent laboratory validation of 

BCS analytical method 00969 for the 

determination of residues of BYI08330-

enol in materials of animal origin 

PTRL Europe, Ulm, Germany 

Bayer CropScience AG, 

Report No.: P613060584, 

Edition Number: M-269571-01-2 

Method Report No.: P/B 964 G 

Date: 20.04.2006 

GLP, unpublished 

Yes BCS 

KIIA 4.4 /01 Brumhard, B. 2006 Analytical Method 00986 for the 

determination of residues of BYI08330 

and BYI08330-enol in soil by HPLCMS/MS 

Bayer CropScience AG, 

Report No.: 00986, 

Edition Number: M-268586-01-1 

Date: 22.03.2006 

GLP, unpublished 

Yes BCS 
– 53 – 

Spirotetramat – Volume 3, Annex B.5, Methods of Analysis April 2008

Annex 

point/ 

reference 

number 

Author(s) Year 

Title 

Source (where different from 

company) 

Company, Report No 

GLP or GEP status (where relevant)

Published or not 

Data 

Protection 

Claimed 

Y/N-R/NR 

Owner 

KIIA 4.5 /01 Brumhard, B. 2006 Analytical method 00836 for the 

determination of BYI08330 and 

BY08330-enol in drinking and surface 

water by HPLC-MS/MS and HPLC-UV 

Bayer CropScience AG, 

Report No.: MR-131/03, 

Edition Number: M-250638-01-2 

Date: 01.08.2006 

GLP, unpublished 

Yes BCS 

KIIA 4.7 /01 Stupp, H. P. 2006 Method for the determination of BYI 

08330 in air by HPLC-UV and 

confirmation by DAD (UV spectra 

match) 

Bayer CropScience AG, 

Report No.: 01015, 

Edition Number: M-274784-02-1 

Method Report No.: MEF-06/119 

Date: 06.06.2006, Amended: 

07.11.2006 

GLP, unpublished 

Yes BCS 

Annex III Data and Information

KIIIA1 5.2.1 

/01 

Zitzmann, W. 2004 Determination of Spirotetramat in

formulations - assay - HPLC, external 

standard 

Bayer CropScience AG, 

Report No.: AM002104MF2, 

Edition Number: M-068817-02-2 

Date: 11.05.2004, Amended: 

26.03.2007 

Non GLP, unpublished 

Yes BCS 

KIIIA1 5.2.1 

/02 

Odendahl, A. 2005 Validation of HPLC-method 

AM002104MF1 - determination of BYI 

08330 in formulations - 

Bayer CropScience AG, 

Report No.: VB2-AM002104MF1, 

Edition Number: M-260838-02-2 

Date: 14.11.2005 

Non GLP, unpublished 

Yes BCS