Two patients died after being treated by a herbalist for mental illness. Samples from the patients and herbal plants were analyzed. Liquid chromatography-mass spectrometry identified several toxic alkaloids, including buphanamin and powelline, in the samples. One of the plants was identified as Boophane disticha, which is known to contain toxic alkaloids. Further work will aim to isolate and characterize compounds from the plant, establish detection limits, and develop the electron impact technology as a screening technique for unknown toxic compounds.

1. Detection of Toxic
Alkaloids by LC-MS
(Electron Impact)
Forensic Toxicology Research Unit
Forensic Chemistry Laboratory, Johannesburg
Department of Health
South Africa
Email: fcljhb@icon.co.za
Department of Health

2. Introduction
 In South Africa extensive use is made of
traditional african medicine
 A large number of the plants used are known
to be toxic
 There is no formal training of herbalists or set
methods for preparing medicines
 There are few analytical methods available
for identifying toxic compounds in these
traditional medicines
 The Forensic Toxicology Research Unit was
formed to develop analytical methods to
identify and screen for toxic herbal
compounds in biological matrices

4.  Two african males from Standerton area were
taken to a herbalist to be treated for “mental
illness”
 Both patients were treated with herbal
enemas
 Both patients became comatose and died
within 3 hours of administration of the enemas
 Viscera samples and plant bulbs were sent to
the Forensic Chemistry Laboratory
Johannesburg for chemical analysis
Case History

5. Sample Preparation
Phosphate-buffered saline added
to homogenized sample
Resultant solution filtered
Filtrate added and drawn through
a Waters Oasis HLB cartridge
Sample eluted from cartridge
with acetonitrile
Evaporated to dryness and
reconstituted with 0.5 mL
methanol

6. HPLC-MS Electron Impact
Identification of unknowns using GC-MS
libraries (NIST, Wiley etc)
Small organic molecules ( 1000
Dalton)
Thermally labile compounds
10
4
10
2
10
1
10
3
10
5
Ionic
Nonpolar
Molecular Weight
Electrospray
APcI
Therma
BeamGC/MS
Application Ranges of MS Techniques
Polar

7. Cold Trap
Expansion
Region
- 5000V
Helium
Fused Silica Capillary
Ion
Volume
Filament
Extractor
Pre Filter Quadrupole Filter
Dynode
Multiplier
Signal
Exit Lens
Ionizer
Mass Filter
Ion Detector
10-5 - 10-6 TORR
2nd
0.5 Torr
225
warm
+ 8V
- 30V - 10V 0 V
- 100V
-800 - -2500V
Gnd.
+ + + + + + +
+ -
-
-
70 eV
Nebulizer
Momentum
Separator
FROM HPLC
1st 5 Torr
80°
65
150
RV18
RV18
Turbo
Pump

9. Instrumental conditions
Pump System Waters Alliance 2690
Mobile phase 95:5 water:ACN,
ramping to 10:90
MeOH:ACN in 35
minutes
Column Phenomenex Aqua C18
250x2mm
Detector(s) Waters 996 PDA
Waters Thermabeam
Mass detector

10. Intensity
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
Minutes
0.00 10.00 20.00 30.00 40.00 50.00 60.00
14.391
15.974
Colon extract @ 301m/z
Intensity
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
301.30
Intensity
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
0.0007
0.0008
0.0009
m/z
50.00 100.00 150.00 200.00 250.00 300.00 350.00
56.05
57.04
91.10
115.14
203.17
217.23
229.30
258.17
301.24

11. NIST match for Buphanamin
14.3922 Bu p h a n a m in
50 70 90 110 130 150 170 190 210 230 250 270 290 310
0
100
100
55 63 77
89
91
102
103
115
121
127
131
141
145 165
165
175
179
190
190
204
204
218
218
231
231
244
244
256
256
272
272
282
282
301
301
Forward Search 879 Reverse Search 952 Probability 90.4%

12. NIST match for Powelline
15.97 01 Po w e llin e
50 70 90 110 130 150 170 190 210 230 250 270 290 310
0
100
100
56
57 67
77
79 91
91
102
102
115
115 127
128
141
153
157
171
189
190 203
203
217
217
229
229
244
246 258
258
272
272
284
284
301
301
Forward Search 809 Reverse Search 811 Probability 76.3%

13. Alkaloids detected in viscera
samples using the TMD
Alkaloid Stomach 1 Liver 1 Kidney 1 Liver 2 Kidney 2 Colon 2
Buphanamin
-     
Buphanisine  - -   
Buphandrin      
Nerbowdin
-3-Acetyl
- - - - - -
Amberline - - - - - -
Powelline
- -  - - 
Chlidanthine,
1,2-dihydro
- - - - - -

14. Identification of bulbs
Papery scales

15. Note growth
pattern

18. HPLC-MS Chromatogram of
Boophane disticha bulb extract
Intensity
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Minutes
0.00 10.00 20.00 30.00 40.00 50.00 60.00
12.402
13.48214.27214.72615.52816.23016.44317.178
18.865
20.784
22.10922.47422.99223.64624.26924.65525.47826.00126.46127.059
28.15328.386
29.33929.991
33.817
35.773
Total Ion
Chromatogram

19. Intensity
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
Minutes
0.00 10.00 20.00 30.00 40.00 50.00 60.00
14.306
15.740
17.177
18.781
Boophane disticha bulb extract @ 301m/z
Intensity
0.00
0.02
0.04
0.06
0.08
138.03 282.18
301.10
Intensity
0.000
0.001
0.002
0.003
0.004
167.91
216.93
229.05
301.06
Intensity
0.00
0.05
0.10
0.15
0.20
m/z
50.00 100.00 150.00 200.00 250.00 300.00 350.00
129.98
215.00
228.04
231.06
245.08
260.18 284.20
300.10
315.10

20. 1 4 .3 0 5 5 B u p h a n a m in
5 0 7 0 9 0 1 1 0 1 3 0 1 5 0 1 7 0 1 9 0 2 1 0 2 3 0 2 5 0 2 7 0 2 9 0 3 1 0
0
1 0 0
1 0 0
5 3 6 3 8 1 8 9
8 9 1 0 2
1 0 9
1 1 5
1 2 7
1 2 7
1 3 8
1 4 1
1 5 4 1 6 5
1 6 5
1 7 8
1 7 9
1 9 0
1 9 0
2 0 4
2 0 4
2 1 8
2 1 8
2 3 1
2 3 1
2 4 2
2 4 4
2 5 6
2 5 6
2 7 2
2 7 2
2 8 2
2 8 2
3 0 1
3 0 1
1 8 .7 7 9 2 B u p h a n d rin
5 0 7 0 9 0 1 1 0 1 3 0 1 5 0 1 7 0 1 9 0 2 1 0 2 3 0 2 5 0 2 7 0 2 9 0 3 1 0 3 3 0
0
1 0 0
1 0 0
5 5
5 5
6 3
6 3
7 1
7 1
8 9
9 1
1 0 1
1 0 1
1 1 5
1 1 5
1 3 0
1 3 0
1 4 1
1 4 1 1 5 3
1 5 9
1 6 5
1 8 7
1 8 7
2 0 2
2 0 2
2 1 5
2 1 5
2 2 8
2 3 1
2 4 5
2 4 5
2 6 0
2 6 0
2 7 2
2 7 2
2 8 4
2 8 4
3 0 0
3 0 0
3 1 5
3 1 5
1 5 .7 3 6 1 P o w e llin e
5 0 7 0 9 0 1 1 0 1 3 0 1 5 0 1 7 0 1 9 0 2 1 0 2 3 0 2 5 0 2 7 0 2 9 0 3 1 0 3 3 0
0
1 0 0
1 0 0
5 6
5 6
7 7 9 1
9 7
1 0 2
1 1 5 1 2 7 1 4 1 1 5 7
1 6 8
1 8 7
1 8 9
2 0 3
2 0 3
2 1 7
2 1 7
2 2 9
2 2 9
2 4 4
2 4 6 2 5 8
2 5 8 2 7 2 2 8 4
3 0 1
3 0 1
3 1 5 3 3 3

21. Compound Detected
(NIST MS Search 2.0 )
Forward
Search
Reverse
Search
%
Probability
Chlidanthine, 1,2-dihydro 674 710 72.7
Buphanamin 867 966 68.4
Crinan-11-ol,1,2-didehydro-
3,7-dimethoxy-{3,11S}
860 867 98.0
Powelline 499 509 63.2
Oxopowelline 726 822 75.2
Buphanisine 874 874 94.5
Buphandrin 910 910 97.5

23. Confirmation HPLC-MS with
GC-TOFMS
Alkaloid HPLC-MS GC-TOFMS
Buphanamin  
Buphanisine  
Buphandrin  
Nerbowdin
-3-Acetyl
 -
Amberline  -
Powelline  
Chlidanthine,
1,2-dihydro
 

24. The way forward
 No commercial standards available to
determine limits of detection (5-30ng on
column?)
 Large volume extract to be prepared &
fractionated using Waters FractionLynx
 Purified fractions to be chemically
characterized using NMR & HPLC-MS
 % Recovery, LOD & LOQ to be established
 Electron Impact technology is compatible with
HPLC and is an excellent screening technique
for unknown compounds

25. Acknowledgements
 TIAFT Organising committee
 Rand Afrikaans University, particularly
Professor B-E van Wyk
Professor F R van Heerden
 Microsep (PTY) LTD
 CSIR
 LECO SA
 Paul Steenkamp
 Department of Health

26. A special thank you to my
wide-eyed audience !