KRA-01

FDA/ORA/ORS

LIB #4578 9 of 25

Data Analysis and Reporting GC/MS Data Analysis

The GC/MS method was adapted from the procedure used by Chan et al [3]. A mitragynine solvent standard was injected at a concentration of 10 µg/mL or 20 µg/mL with a corresponding retention time of 14.90 minutes. A 7-hydroxy mitragynine standard was acquired to determine the retention time of this alkaloid and has a corresponding retention times of 13.46 minutes. Figure 2 is an example of a mitragynine and 7-hydroxy mitragynine solvent standard and the associated full scan mass spectrum. The solvent standards retention time and mass spectrum was used for confirmation of mitragynine in the variety of products received by the Denver Laboratory. Figure 3 is an example of the QEDIT report generated for the reporting of data. Figure 3 thru 5 are examples of a negative control, positive control, and a powder leaf sample. Each figure included the total ion chromatogram (TIC), mass spectrum, and an example of the QEDIT report. With the GC/MS method, Chromatography/Mass Spectrometry guidance CVM118 [5] for MS 1 full scan data was used to determine confirmation of identity along with retention time matching, 2% GC/MS relative to retention time of the standard and signal–to-noise (s/n) threshold of 3:1. The mass spectrum of the mitragynine standard was compared to the WILEY library and demonstrated a match of greater than 98%. For mass spectral confirmation of mitragynine in the samples analyzed by GC/MS, the mitragynine solvent standard was compared to the matching peak in the chromatogram of the sample. Mitragynine is confirmed as positive if the mass spectrum of the sample matches the spectrum of the mitragynine solvent standard. Another aspect of ensuring the GC/MS system was suitable for anlysis was performing a visual inspection of the postive control sample. The GC/MS demonstrated a consistent pattern with mitragynine peak eluting first and two other peak eluting at 15.02 minutes and 15.20 minutes, Figure 6. The TIC aquired from the sample was similar to the GC/MS TIC from reference 3. Hence, beside the presence of mitragynine, these two other peaks have been noted with the postive control and all the sample anlyzed at the Denver Laboratory. Sample that are positive via the GC/MS qualitative screening method are then analyzed by the UPLC/PDA or LC-MS/MS for quantitation.