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BCS-RD-D-ROCS Method-No. 00857
D-40789 Monheim Report No. MR-099/04
1 Summary
The analytical method 00857 presented in this report was developed in order to
determine residues of BYI08330 (parent compound, total residue of BYI08330) and its
metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and
BYI08330-enol-Glc in/on plant material by HPLC-ESI-MS/MS using stable isotopically
labelled internal standards.
BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-monohydroxy
and BYI08330-enol-Glc are extracted from the sample material using a blender
with an acidic acetonitrile/water mixture (4/1, v/v). After filtration of the extract, the
corresponding stable isotopically labelled analytes are added. Afterwards, the solution is
made up to volume, diluted and subjected to HPLC-MS/MS without any further clean-up
step (with exception of hop where a second filtration step was used).
For quantification external calibration with standard solutions quoting the peak area ratio
of the analytes and the corresponding stable isotopically labelled internal standards
were used. If matrix problems are observed, HPLC-MS/MS conditions have to be
adapted.
The limit of quantitation (LOQ) is 0.01 mg/kg for each single compound corresponding
to the lowest fortification level of successfully conducted recovery experiments with
exception of hop cone with a LOQ of 0.10 mg/kg. The limit of detection (LOD) for all
single compounds was estimated to be at about 4 times lower than the corresponding
LOQ, as could be concluded from the linearity data and matrix interference in untreated
control samples. For determination of the total residue of BYI08330 (sum of single
analyte residues each calculated as BYI08330 equivalents), the LOQ is calculated as
0.055 mg/kg for all matrices with exception of hop cone (0.55 mg/kg).
A linear correlation between the injected amount of the analytes and the detector
response was observed for standards in solvent (5 ng/L to 50 µg/L) using stable
isotopically labelled internal standards. The correlation coefficients of the 1/x weighted
linear regression were 0.9976 to 0.9995.
The occurrence of matrix effects was not monitored during method development and
validation, as the internal standard procedure using stable isotopically labelled internal
standards compensates for matrix effects. If matrix interferences or matrix effects are
observed, the HPLC-MS/MS conditions have to be adapted.
The stability of BYI08330 and its metabolites BYI08330-enol, BYI08330-ketohydroxy,
BYI08330-mono-hydroxy and BYI08330-enol-Glc in stock and standard solutions
(water/acetonitrile, 2/8, v/v + 0.22 mL/L formic acid) was tested.
The results indicate that all analytes are stable in standard solutions for at least four
weeks of storage in a refrigerator at 4°C ± 3°C under dark conditions.
Blank values in untreated control samples were well below 30% of the LOQ.