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

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Bayer CropScience AG Page 34 of 82 BCS-RD-D-ROCS Method-No. 00857 D-40789 Monheim Report No. MR-099/04 5.3 Linearity of the Detector (contd) The occurrence of matrix effects was not monitored during method development and validation, as the internal standard procedure using stable isotopically labelled internal standards compensates for matrix effects. The calibration data obtained justify using the single point calibration method for calculation of the residues of BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-Glc. However, an appropriate bracketing standard concentration, corresponding to the order of magnitude of the residues should be used for quantification and it should be noted that the concentration of the stable isotopically labelled internal standard in the calibration and sample solutions must be kept at the same level. The linearity of the detector used was tested for all HPLC conditions given in chapter 4.3.2 to 0. The corresponding MS parameters are given in chapter 4.3.6. 5.4 LOQ, LOD, Recovery Rates (Accuracy) and Precision (Repeatability) Recovery rates were determined at fortification levels of 0.01 mg/kg (= LOQ level), and 1.0 mg/kg (each compound). For hop cone (dried and green) fortification levels of 0.10 mg/kg (= LOQ level) and 10 mg/kg (each compound) were validated. The lowest fortification level experimentally providing a mean recovery between 70 and 110% with a relative standard deviation of ≤ 20% per definition corresponds to the Limit of Quantitation (LOQ), provided that blank values are below 30% of this level. The detection limit (LOD) was estimated to be at least 4 times lower than the LOQ. Recovery experiments were conducted by separate fortification of untreated control samples with defined amounts of BYI08330 or its metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-Glc prior to analysis. Results are presented in the following tables. For chromatograms see Appendix 1. The individual recovery rates for BYI08330 ranged from 72 to 108% with overall means (n = 6 - 10) between 87 and 101% and overall RSDs of up to 13.3%. The individual recovery rates for BYI08330-enol ranged from 69 to 111% with overall means (n = 6 - 10) between 86 and 104% and overall RSDs of up to 15.0%. The individual recovery rates for BYI08330-ketohydroxy ranged from 69 to 111% with overall means (n = 6 - 10) between 87 and 101% and overall RSDs of up to 14.6%. The individual recovery rates for BYI08330-mono-hydroxy ranged from 68 to 110% with overall means (n = 6 - 10) between 88 and 101% and overall RSDs of up to 13.7%. The individual recovery rates for BYI08330-enol-Glc ranged from 70 to 109% with overall means (n = 6 - 10) between 86 and 98% and overall RSDs of up to 15.1%. As a measure for the precision of the method, the intra-laboratory repeatability (n = 5) is given as relative standard deviation (% RSD) for different sample materials (e.g. orange fruit, plum fruit, apple fruit, apple sauce, potato tuber, hop beer) at fortification levels of 0.01 and 0.1 mg/kg or 1.0 and 10 mg/kg (only hop cone dried and green). The RSD of the repeatability tests at each recovery set ranged from 0.5 to 14.0%. All method validation data are in compliance with the guideline requirements for residue data generation [4].
Bayer CropScience AG Page 35 of 82 BCS-RD-D-ROCS Method-No. 00857 D-40789 Monheim Report No. MR-099/04 5.4 LOQ, LOD, Recovery Rates and Repeatability (contd) Table 12: Recoveries for BYI08330; RSD: Relative Standard Deviation. Crop Fortification Level (FL) (mg/kg) Recoveries % (Single Values) Mean (%) per FL RSD (%) Mean (%) overall RSD (%) overall Orange 0.01 95 100 75 90 77 87 12.6 Fruit 1.0 87 86 95 92 92 90 4.2 89 8.9 Orange 0.01 90 91 93 91 1.7 Juice 1.0 68 88 92 83 15.6 87 10.9 Orange 0.01 106 104 97 102 4.6 Pomace 1.0 94 85 73 84 12.5 93 13.3 Orange 0.01 102 106 107 105 2.5 Jam 1.0 96 97 99 97 1.6 101 4.6 Sweet Cherry 0.01 100 99 87 95 7.6 Fruit 1.0 91 91 88 90 1.9 93 6.0 Plum 0.01 100 91 99 90 104 97 6.3 Fruit 1.0 96 87 85 83 91 88 5.9 93 7.5 Peach 0.01 89 91 102 94 7.4 Fruit 1.0 91 92 94 92 1.7 93 5.0 Strawberry 0.01 96 97 92 95 2.8 Fruit 1.0 86 94 95 92 5.4 93 4.3 Apple 0.01 84 92 95 73 93 87 10.4 Fruit 1.0 87 86 90 85 91 88 2.9 88 7.2 Apple 0.01 96 94 94 95 1.2 Pomace Dried 1.0 89 89 91 90 1.3 92 3.2 Apple 0.01 98 91 91 99 95 95 4.0 Sauce 1.0 88 84 86 86 88 86 1.9 91 5.8 Tomato 0.01 100 102 102 98 108 102 3.7 Fruit 1.0 100 97 100 98 98 99 1.4 100 3.2 Tomato 0.01 93 100 100 97 98 98 3.0 Juice 1.0 92 93 95 95 97 94 2.1 96 3.0 Tomato 0.01 97 100 89 95 6.0 Preserve 1.0 90 91 92 91 1.1 93 4.7 Grape 0.01 93 104 95 93 98 97 4.8 Bunches of Grape 1.0 90 90 92 90 91 91 1.0 94 4.8 Grape 0.01 98 97 98 98 0.6 Most 1.0 92 94 93 93 1.1 95 2.8 Grape 0.01 89 97 97 94 4.9 Pomace 1.0 89 87 90 89 1.7 92 4.8 Grape 0.01 99 101 95 98 3.1 Wine 1.0 93 94 92 93 1.1 96 3.7 Potato 0.01 91 95 97 90 91 93 3.3 Tuber 1.0 91 90 90 87 87 89 2.1 91 3.4 Melon 0.01 97 99 95 97 2.1 Peel 1.0 87 88 93 89 3.6 93 5.2 Melon 0.01 90 93 97 98 94 94 3.4 Pulp 1.0 86 85 90 90 92 89 3.3 92 4.6 Onion 0.01 91 96 95 97 95 95 2.4 Tuber 1.0 94 91 89 91 89 91 2.3 93 3.2 Hop 0.01 108 92 75 89 99 93 13.2 Beer 1.0 92 89 86 87 90 89 2.7 91 9.4 Hop 0.10 94 97 99 97 2.6 Cone Dried 10.0 93 87 87 89 3.9 93 5.4 Hop 0.10 86 95 100 94 7.6 Cone Green 10.0 82 88 84 85 3.6 89 7.8 Broccoli 0.01 95 104 104 107 87 99 8.3 Curd 1.0 76 96 81 79 100 86 12.5 93 12.2 Cauliflower 0.01 87 94 100 94 6.9 Inflorescence 1.0 93 72 92 86 13.8 90 10.7 Lettuce 0.01 95 81 97 91 9.6 Head 1.0 101 97 86 95 8.2 93 8.2
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Bayer CropScience AG Page 1 of 82 BCS-RD-D ... - IR-4 Project

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