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Bayer CropScience AG Page 22 of 82 BCS-RD-D-ROCS Method-No. 00857 D-40789 Monheim Report No. MR-099/04 4.2 Analytical Method Extraction Procedure 1. Weigh a representative aliquot of 10 g of the sample into a 150-mL beaker. Remark: Do not allow deep-frozen sample material to thaw before extraction. Recovery samples have to be extracted immediately after spiking. 2. Add 40 mL (60 mL for hop cone material) of acetonitrile/water (4/1, v/v, containing 0.22 mL formic acid/L) to the sample. 3. Blend the sample some minutes with a Polytron blender. 4. Rinse the blender with approx. 5 mL of acetonitrile/water (4/1, v/v, containing 0.22 mL formic acid/L). 5. Mount a syringe tube in working position on top of a VacElut-Station and place a BondElut clean polyethylene frit onto the bottom of the syringe tube. 6. Add 2.5 g of Celite 545 to the sample and filter the content of the beaker carefully through the syringe tube using low vacuum. Collect the extract in a 100-mL volumetric flask. 7. Wash the filtered solids with a total of 30 mL of acetonitrile/water (4/1, v/v, containing 0.22 mL formic acid/L) and discard the filtered solids. Add 1 mL of internal standard mixture (100 µg/L of each compound in acetonitrile/water, 2/8, v/v, containing 0.22 mL formic acid/L) and make up to volume with acetonitrile/water (4/1, v/v, containing 0.22 mL formic acid/L). 8. Transfer an aliquot of 0.1 mL into a 1.8-mL HPLC sample vial, add a volume of 0.9 mL internal standard mixture (containing 1 µg/L of each 13 C3-labelled compound in acetonitrile/water, 15/85, v/v, containing 0.22 mL formic acid/L) and subject to liquid chromatography and MS/MS determination. 9. For hop material (cone dry, green): transfer an aliquot of 0.1 mL of (7) to a 10-mL volumetric flask and make up to volume with internal standard mixture (containing 1 µg/L of each 13 C3-labelled compound in acetonitrile/water (15/85, v/v, containing 0.22 mL formic acid/L). Filter through a 0.45 µm single use filter (Sartorius Minisart RC15, order no. 17762) and subject to liquid chromatography and MS/MS determination.
Bayer CropScience AG Page 23 of 82 BCS-RD-D-ROCS Method-No. 00857 D-40789 Monheim Report No. MR-099/04 4.3 Analysis by HPLC-MS/MS 4.3.1 Principle of Measurement An aliquot of the prepared sample solution is injected into the high performance liquid chromatograph and subjected to reversed phase chromatography coupled with tandem mass spectrometry with electrospray ionization. The protonated molecular ions of the analytes are accelerated by the adequate voltage regulation and filtered by the first quadrupole due to its mass-to-charge (m/z) ratio. These precursor ions are impulsed with nitrogen in the collision cell (second quadrupole) and each fragment ion (product ions) is separated according to its m/z ratio in the third quadrupole, two of these product ions per analyte are selected for multiple reaction monitoring (MRM) one product ion (MRM-transition) serving for quantitation and the second for confirmation. 4.3.2 HPLC Conditions for BYI08330 and BYI08330-enol Instrument: e.g. Agilent 1100 Injector: e.g. HTC PAL, CTC Analytics Column: e.g. Agilent Zorbax Eclipse-XDB-C8, 5 µm, 150 x 4.6 mm i.d. with pre-column or equivalent Injection Volume: e.g. 10 µL Oven temperature: 40°C Mobile Phase: Bin Pump A: Water/acetonitrile (9/1, v/v), containing 0.22 mL/L formic acid) Bin Pump B: Acetonitrile, containing 0.22 mL/L formic acid Iso Pump C: Water/acetonitrile (1/1, v/v), containing 0.1 mL/L acetic acid or formic acid) Time Table: Time [min] A [%, v/v] B [%, v/v] Into MS Into Waste 0.0 60 40 Iso pump Bin pump 0.5 60 40 Iso pump Bin pump 2.0 Bin pump Iso pump 3.5 10 90 4.5 10 90 4.6 60 40 5.0 Iso pump Bin pump 6.0 60 40 6.0 Stop time Flow (Column): 1.0 mL/min Flow (into MS): 1.0 mL/min Retention times: BYI08330-enol approx. 2.7 min BYI08330-enol 13 C3 approx. 2.7 min BYI08330 approx. 4.3 min BYI08330 13 C3 approx. 4.3 min
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