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BCS-RD-D-ROCS Method-No. 00857
D-40789 Monheim Report No. MR-099/04
Table of Contents Page
Study Title .................................................................................................................................................. 1
Data Confidentiality Statement .................................................................................................................. 2
Certification of Good Laboratory Practice.................................................................................................. 3
Certification of Authenticity ........................................................................................................................ 4
Quality Assurance Statement .................................................................................................................... 5
Sponsor...................................................................................................................................................... 6
Laboratories Involved in the Study............................................................................................................. 6
Schedule .................................................................................................................................................... 6
Archiving .................................................................................................................................................... 6
1 Summary ........................................................................................................................................... 9
2 Introduction...................................................................................................................................... 11
2.1 BYI08330 ................................................................................................................................ 11
2.2 BYI08330-enol ........................................................................................................................ 11
2.3 BYI08330-ketohydroxy ........................................................................................................... 12
2.4 BYI08330-mono-hydroxy........................................................................................................ 12
2.5 BYI08330-enol-Glc ................................................................................................................. 12
2.6 BYI08330 13 C3 (FHN 12952)................................................................................................... 13
2.7 BYI08330-enol 13 C3 (FHN 13777) .......................................................................................... 13
2.8 BYI08330-ketohydroxy 13 C3 (FHN 14066) .............................................................................. 13
2.9 BYI08330-mono-hydroxy 13 C3 (FHN 15436)........................................................................... 14
2.10 BYI08330-enol-Glc 13 C3 (KTS 10115)..................................................................................... 14
3 Sampling and Preparation of Laboratory Samples ......................................................................... 15
4 Experimental Section ...................................................................................................................... 15
4.1 Materials ................................................................................................................................. 16
4.1.1 Apparatus ........................................................................................................................... 16
4.1.2 Reagents/Supplies ............................................................................................................. 16
4.1.3 Reference Items .................................................................................................................17
4.1.4 Stock Solutions................................................................................................................... 18
4.1.5 Standard Solutions ............................................................................................................. 19
4.1.6 Standard Solutions of Stable-Labelled Compounds .......................................................... 20
4.1.7 Standard Mixture Solutions ................................................................................................ 21
4.2 Analytical Method ................................................................................................................... 22
4.3 Analysis by HPLC-MS/MS...................................................................................................... 23
4.3.1 Principle of Measurement .................................................................................................. 23
4.3.2 HPLC Conditions for BYI08330 and BYI08330-enol.......................................................... 23
4.3.3 HPLC Conditions for BYI08330-ketohydroxy, BYI08330-mono-hydroxy and
BYI08330-enol-Glc ............................................................................................................. 24
4.3.4 HPLC Conditions BYI08330-enol-Glc (Individual Determination)...................................... 25
4.3.5 HPLC Conditions BYI08330 (Individual Determination) .................................................... 26
4.3.6 MS/MS Conditions.............................................................................................................. 27
4.3.7 Analytical Procedure .......................................................................................................... 28
4.4 Calculation .............................................................................................................................. 29
4.4.1 Calculation of the Residues ............................................................................................... 30
4.4.2 Calculation of Recovery Rates........................................................................................... 30
4.4.3 Calculation of Total Residue of BYI08330 ......................................................................... 31
5 Results and Discussion................................................................................................................... 32
5.1 Selectivity................................................................................................................................ 32
5.2 Blank Values of Untreated Control Samples .......................................................................... 32
5.3 Linearity of the Detector ......................................................................................................... 33
5.4 LOQ, LOD, Recovery Rates and Repeatability...................................................................... 34
5.5 Stability of BYI08330 and its Metabolites in Plant Extracts.................................................... 40
5.6 Stability in Standard and Stock Solution................................................................................. 42
6 References ...................................................................................................................................... 42