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

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

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

Sample PreparationPhosphate-buffered saline added

to homogenized sampleResultant solution filteredFiltrate added and drawn through

a Waters Oasis HLB cartridgeSample eluted from cartridge

with acetonitrile Evaporated to dryness and

reconstituted with 0.5 mL methanol

HPLC-MS Electron Impact

Identification of unknowns using GC-MS libraries (NIST, Wiley etc)

Small organic molecules ( 1000 Dalton)

Thermally labile compounds

104

102

101

103

105

Ionic

Nonpolar

Molecular Weight

Electrospray

APcI

ThermaBeamGC/MS

Application Ranges of MS Techniques

Polar

Cold Trap

Expansion Region

- 5000V

HeliumFused Silica Capillary

IonVolume

Filament

Extractor

Pre Filter Quadrupole FilterDynode

Multiplier

Signal

Exit Lens

IonizerMass Filter Ion Detector

10-5 - 10-6 TORR

2nd0.5 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

Instrumental conditionsPump 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 PDAWaters Thermabeam Mass detector

Inte

nsity

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

14.3

9115

.974

Colon extract @ 301m/z

Inte

nsity

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035301.30

Inte

nsity

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

56.0557.04

91.10115.14

203.17

217.23

229.30

258.17

301.24

NIST match for Buphanamin

1 4 . 3 9 2 2 B u p h a n a m i n

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 5 6 3 7 7

8 9

9 1

1 0 2

1 0 3

1 1 5

1 2 1

1 2 7

1 3 1

1 4 1

1 4 5 1 6 5

1 6 5

1 7 5

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 4

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

Forward Search 879 Reverse Search 952 Probability 90.4%

NIST match for Powelline

1 5 . 9 7 0 1 P o w e l l i n 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

0

1 0 0

1 0 05 6

5 7 6 7

7 7

7 9 9 1

9 1

1 0 2

1 0 2

1 1 5

1 1 5 1 2 7

1 2 8

1 4 1

1 5 3

1 5 7

1 7 1

1 8 9

1 9 0 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 7 2

2 8 4

2 8 4

3 0 1

3 0 1

Forward Search 809 Reverse Search 811 Probability 76.3%

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

- - - - - -

Identification of bulbs

Papery scales

Note growth pattern

HPLC-MS Chromatogram of Boophane disticha bulb extract

Inte

nsity

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

12.4

0213

.482

14.2

7214

.726

15.5

2816

.230

16.4

4317

.178

18.8

6520

.784

22.1

0922

.474

22.9

9223

.646

24.2

6924

.655

25.4

7826

.001

26.4

6127

.059

28.1

5328

.386 29

.339

29.9

91

33.8

1735

.773

Total Ion Chromatogram

Inte

nsi

ty

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

Minutes0.00 10.00 20.00 30.00 40.00 50.00 60.00

14

.30

615

.74

017

.17

71

8.7

81

Boophane disticha bulb extract @ 301m/z

Inte

nsity

0.00

0.02

0.04

0.06

0.08

138.03 282.18

301.10

Inte

nsity

0.000

0.001

0.002

0.003

0.004

167.91

216.93

229.05

301.06

Inte

nsity

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

129.98215.00

228.04

231.06

245.08

260.18 284.20300.10

315.10

1 4 . 3 0 5 5 B u p h a n a m i n

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 r i n

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 l l i n 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 67 7 9 1

9 7

1 0 21 1 5 1 2 7 1 4 1 1 5 7

1 6 81 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

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

Confirmation HPLC-MS with GC-TOFMS

Alkaloid HPLC-MS GC-TOFMS

Buphanamin

Buphanisine

Buphandrin

Nerbowdin-3-Acetyl

-

Amberline -

Powelline

Chlidanthine, 1,2-dihydro

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

Acknowledgements

TIAFT Organising committee Rand Afrikaans University, particularly

Professor B-E van WykProfessor F R van Heerden

Microsep (PTY) LTD CSIR LECO SA Paul Steenkamp

Department of Health

A special thank you to my wide-eyed audience !