KRA-01

FDA/ORA/ORS

LIB #4578 23 of 25

mitragynine. Validation was performed by characterizing a dry leaf Kratom product and extracting this material seven times a day over a three day period for a total number of extraction n=21, refer to Table 4. Once the positive control was characterized, the same positive control was extracted in duplicate (n=2) for every analytical batch. The UPLC/PDA data demonstrated a mean value of 1.041% (n=21, 4.2%) and the LC-MS/MS 1.140% (n=14, 6.81%) for mitragynine in the positive control. Table 4 : Validation Data of Mitragynine in Positive Control Day Mean % (RSD) Mean % (RSD) UPLC LC-MS/MS 1 (n=7) 1.061 (3.6%) 1.156 (5.95%) 2 (n=7) 1.056 (3.1%) 1.125 (7.82%) 3 (n=7) 1.004 (4.4%) n/a Overall (n=21, 14) 1.041 (4.2%) 1.1403 (6.81%) The same positive control was used for all the analysis of products received by the Denver Laboratory. Table 5 is a summary of three different chemists extracting the positive control over a 1 year time frame and analyzed on the UPLC/PDA. The overall results demonstrate the extraction procedure generates reproducible results and the positive control is homogenous.

Table 5: Positive Control over 2 years and different analysis

Batches

Analyst Control

% Mitragynine

1

A

Positive Control

1.177 1.110 1.182 1.110 1.092 1.321 1.241 1.207 1.101 1.131

Positive Control Duplicate

2

A

Positive Control

Positive Control Duplicate

3

B

Positive Control

Positive Control Duplicate

4

B

Positive Control

Positive Control Duplicate

5

C

Positive Control

Positive Control Duplicate

Mean ( n=10)

1.1672 0.074

sd

%RSD

6.31

The same set of samples were extracted and analyzed via both the UPLC/PDA and the LC- MS/MS. The results for the amount of mitragynine are similar; hence the LC-MS/MS method can be used for the confirmation and quantitation of mitragynine in Kratom.