KRA-01

FDA/ORA/ORS

LIB #4578 22 of 25

Figure 18: a) Sample Dried Powder Leafs - Total Ion Chromatogram (TIC)

b) Sample Dried Powder Leafs - Reconstructed Ion Chromatogram (RIC)

NL: 3.70E6

Kraton_28Dec12_A028 - TIC - SM: 5 RT: 5.00 - 14.99

RT: 11.51

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Intensity

a

RT: 12.27

11.88

RT: 12.80

0 5

13.50

RT: 14.47

5.41 6.16 6.70

7.25

7.71

8.17

8.70

9.18

9.79

10.60

14

13

11

12

9

10

8

6

7

Time (min)

RT: 10.50 - 15.00

RT: 10.50 - 15.00

NL: 4.83E5 TIC F: + c ESI SRM ms2 399.200 [159.099-159.101, 174.099-174.101, 238.199-238.201] MS ICIS kraton_28dec12_a19

RT: 11.53

NL: 2.17E5 m/z= 173.90-174.10 F: + c ESI SRM ms2 399.200 [159.099-159.101, 174.099-174.101, 238.199-238.201] MS ICIS kraton_28dec12_a19

RT: 11.53

SRM 399.2-> 174 Sample Mitragynine Rt=11.53

100

10 20 30 40 50 60 70 80 90 100

b

95

90

85

Total Ion Chromatogram (TIC) Sample Mitragynine Rt=11.53

RT: 12.27

80

75

RT: 12.82

70

RT: 13.46

10 20 30 40 50 60 70 80 90 100 Relative Abundance 0

NL: 1.35E5 m/z= 159.00-159.20 F: + c ESI SRM ms2 399.200 [159.099-159.101, 174.099-174.101, 238.199-238.201] MS ICIS kraton_28dec12_a19

RT: 11.53

SRM 399.2-> 159.1 Sample Mitragynine Rt=11.53

65

60

55

50

RT: 12.29

45

Relative Abundance

40

RT: 12.27

RT: 12.80

35

0 10 20 30 40 50 60 70 80 90 100 0

NL: 1.31E5 m/z= 238.00-238.20 F: + c ESI SRM ms2 399.200 [159.099-159.101, 174.099-174.101, 238.199-238.201] MS ICIS kraton_28dec12_a19

RT: 11.53

30

SRM 399.2-> 238.1 Sample Mitragynine Rt=11.53

25

20

15

RT: 12.27

10

RT: 12.82

5

RT: 12.80

RT: 13.27

0

11

12

13

14

15

11

12

13

14

15

Time (min)

Time (min)

Method Validation UPLC/PDA and LC-MS/MS

Mitragynine standard was used for the identification, confirmation and quantitation for the determination of Kratom. Due to the low concentration of the mitragynine stock standard (100 µg/mL) and the high level of mitragynine in the samples, traditional spiking of the standard via a wet/dry spike into a negative control was not feasible. Different types of commercial products that claimed to contain Kratom were purchased via the Internet in 2012 and analyzed for