1. Biotoxin analysis in Australia –
Two years on
18 October 2014
Andrew Bradbury
Director, Advanced Analytical Australia

2. Outline
• Background
• Marine toxins
• PSP, ASP & DSP analysis
• Advances in instrumentation
• Fast PSP analysis
• TAT report
• Improvements
• PSP profiling, accumulation & degradation

3. Who is Advanced Analytical?
• Advanced Analytical is an Australian-owned, private and independent
contract testing laboratory
• Established in 2003
• Located in Sydney (laboratory), Brisbane, Perth and Melbourne
• Employ over 40 staff
• Multi-disciplinary laboratory group specialising in organic and inorganic
chemical, microbiological and genetic detection analysis to the
environmental, food, pharmaceutical and agrichemical industries
• Appointed Analyst on FSANZ Laboratory Panel
• Appointed Analyst for AQIS Imported Food Inspection Program
• Successful tenderer for Australian Seafood Biotoxin Partnership (ASBP)

4. Toxic algae
Biotoxin accumulation
Shellfish/Finfish/Crustaceans…….
Fish deaths Farm & recreational closures Human Poisoning

5. Background
• The CRC Seafood review of 2011/2012 identified that there were significant gaps
in biotoxin testing capability in Australia and that there was no laboratory capable
of testing shellfish samples for a wide range of toxins using sophisticated
instrumental techniques.
• At Advanced Analytical, development of methods for biotoxin analysis
commenced in late 2011/early 2012, NATA accreditation was achieved by June
2012
• We successfully tendered for the ASBP (Australian Shellfish Biotoxin Partnership)
contract and testing commenced in July 2012.
• Australia New Zealand Food Standards Code limits
– Paralytic shellfish poisons PSP (Saxitoxin equivalent) – 0.8 ppm
– Amnesic shellfish poisons ASP (Domoic acid equivalent) – 20 ppm
– Diarrhetic shellfish poisons DSP (Okadaic acid equivalent) – 0.2 ppm
– Neurotoxic shellfish poisons NSP – 0.8 ppm

6. Marine Biotoxins - why test for them
• Analysis of PSPs was performed using HPLC with Fluorescence detection (AOAC
2005.06 method) otherwise known as the Lawrence Pre-column oxidation
method
• Lipophilic analysis by LCMSMS is based on the JAOAC 2011, Villar-Gonzalez et al
• Reason for choosing to set up instrumental methods
– Provide more information on toxin profiles than historical methods
– Increased availability of chemical standards
– Improved methods for faster and more sensitive instrumental techniques
• Regulatory drivers
– Official standard reference method (EC No. 2074/2005) in the EU for lipophilic
biotoxins has been the mouse bioassay (MBA)
– MBA is now considered by European Food Safety Authority (EFSA) to be deficient due
to high variability in results, insufficient detection capability and limited specificity
– Acceptance of data from instrumental methods

7. PSP, ASP, DSP Toxins
• PSPs by LC-FLD
– STX
– dcSTX
– GTX1,4
– GTX2,3
– dcGTX2,3
– GTX5
– NEO
– dcNEO
– C1,2
– C3,4
– doSTX
• Lipophilics by LCMSMS
– DA
– OA
– DTX 1 & 2
– PTX 1 & 2
– AZA 1, 2, 3
– YTX
– Homo YTX
– 45 OH YTX
– 45 OH homo YTX
– SPX 1
– GYM
– CYA

9. Chromatograph for PSP toxins
PSP screen: 14 min vs 40 min (HPLC) per sample
Full PSP confirmation: 56 min vs 160 min (HPLC) per sample
TIC of lipophilic toxins spiked on a blank scallop sample (10 ugkg-1)
GTX5
Shellfish matrix
dcGTX2,3
GTX1,4
C3,4
dcGTX2,3
C1,2
NEO
dcSTX
dcNEO
dcSTX
NEO
GTX2,3
GTX1,4
STX
NEO

10. 2013 Ferrari 458 Italia F1 DCT
• $579,900
• 7-Speed Sports Automatic Dual
Clutch
• 8 cylinder 4.5L engine
• 0-100km/h in 3.4 seconds
AB SCIEX QTRAP 5500 LCMSMS
• $586,000
• Linear Accelerator
• 20,000 Da/sec scan speeds
• 100-fold gain in sensitivity in ion
trap scan modes
• 0-200 analytes in 15 minutes

11. The LCMSMS process
• Liquid Chromatography - Mass Spectrometry Mass Spectrometry (LCMSMS), also
known as Triple Quadrupole Mass Spectrometry or Tandem Mass Spectrometry
• Liquid chromatography separates the compounds chromatographically using a
liquid mobile phase before introduction into the mass spectrometer.
• The mass spectrometer ionizes the target chemical compounds to generate
charged molecules or molecule fragments and measuring their mass-to-charge
ratios.
• In MSMS we use a process called ‘Multiple Reaction Monitoring’ (MRM) to isolate
a precursor ion which is further dissociated to product ions. Under controlled
conditions, this provides a unique pattern for each compound.
• In ‘Triple quad’ MS, the combination of unique product ions (providing greater
specificity) and elimination of the background noise results in consistently low
limits of detection in complex matrices.

12. TIC of lipophilic toxins spiked on a blank OYSTER sample (10 ugkg-1)
Expanded +MRM of lipophilic spiked oyster sample
GYM
DA
SPX
PTX-2
AZA-1
AZA-2
AZA-3
OA
YTX
Expanded -MRM of lipophilic spiked oyster sample
DTX-2
DTX-1
Multi-toxin Screen by LCMSMS

13. Now
UPLCs coupled with MS/MS or Fluorescence detector
(Running time < 8 min per injection)

14. Tasmanian Turnaround Time (TAT) Report
Year # Samples Ave. days % Met
2012 218 3.9 74%
2013 640 3.1 95%
2014* 680 2.3 99%
* Jan-Sept

15. Things that impact on TAT
• There are some areas that we can’t always control and these can still have a
major impact on reporting of results on time
• Delays in clients sending samples
– Due to regional and distance issues
– Weather
• Couriers
– Delays in receiving samples
• Instrumentation
– Unexpected breakdowns
• Staffing in laboratory
– Staff on leave
• Sample confirmations

16. Improvements over 2+ years
 Actively cross-trained staff in biotoxin analysis
 Two fully trained analysts who can run all biotoxin analyses on all instruments
 Continuously optimised methods to make process more efficient => faster TAT
 Dedicated instrument for PSP analysis plus 2nd one as backup
 In the final stages of method development and validation of PSP analysis on UHPLC
=> run time reduction of 70%, improved peak shape, shorter TAT in peak periods
 Inclusion of new PSP toxin, deoxydecarbamoyl-saxitoxin (doSTX), into method.
 Have purchased the latest instrument - ABSCIEX 6500 ULCMSMS => improved
sensitivity and processing power
 Sourced Brevetoxin CRMs so can now offer a Brevetoxin (NSP) screen
 Streamlined registration & prioritisation of shellfish samples into the lab
 Agreed communication times and processes with TSQAP

17. Biotoxin detections in Australia – past 2 years
DSP
ASP
PSP
PSP
ASP
DSP
DSP
ASP
PSP
DSP
ASP
PSP
DSP
ASP
PSP

18. 8000
7000
6000
5000
4000
3000
2000
1000
0
18
16
14
12
10
8
6
4
2
0
15-Oct-12 15-Jan-13 15-Apr-13 15-Jul-13 15-Oct-13 15-Jan-14 15-Apr-14 15-Jul-14
(cels per litre)
Algal cel concentration
PSP toxins
(mg per kg STX eq)
Date
PSP Screen
PSP Confirmation
Prewarning level 0.4 mg kg-1
Gymnodinium_catenatum
Port Esperance Lease 192

19. dcGTX2,3
GTX1,4
High C3,4
Periodate
Oxidation
dcSTX, No NEO in POX Peroxide
GTX5
C1,2
Low
GTX2,3
STX
C1,2
dcGTX2,3
GTX5 STX
dcSTX
or NEO
Low
GTX2,3
Oxidation
PSP in Gymnodium bloom

20. PSP in Alexandrium blooms
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
7
6
5
4
3
2
1
0
15-Oct-12 15-Jan-13 15-Apr-13 15-Jul-13 15-Oct-13 15-Jan-14 15-Apr-14 15-Jul-14
(cels per litre)
Algal cel concentration
PSP toxins
(mg per kg STX eq)
Date
PSP Screen
PSP Confirmation
Prewarning level 0.4 mg kg-1
Alexandrium tamarense
Spring Bay Lease 164

21. dcGTX2,3
High GTX1,4
No C3,4
High
GTX2,3
High C1,2
Very low
GTX5
STX
dcSTX
or NEO
High
GTX2,3
High C1,2
dcGTX2,3
Periodate
Oxidation
Peroxide
Oxidation

22. Fast accumulation and degradation of PSP
in shellfish
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
PSP toxins
(mg per kg STX eq) Date
PSP Screen
PSP Confirmation
Prewarning level 0.4 mg kg-1
Negative leve 0.025 mg kg-1
No algal data during the period
Little Swan Port Lease 86

23. Acknowledgement:
Tasmanian Shellfish Quality Assurance Program – Megan, Jason & Howel
Advanced Analytical Biotoxin team – Rama, Feng & Dave
Andrew Bradbury
Advanced Analytical Australia
Ph: 07 3268 1228
andrew.bradbury@advancedanalytical.com.au