Biotoxin analysis in Australia – Two years on

18 October 2014

Andrew Bradbury

Director, Advanced Analytical Australia

Outline

• Background

• Marine toxins

• PSP, ASP & DSP analysis

• Advances in instrumentation

• Fast PSP analysis

• TAT report

• Improvements

• PSP profiling, accumulation & degradation

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)

Toxic algae

Shellfish/Finfish/Crustaceans…….

Biotoxin accumulation

Fish deaths Farm & recreational closures Human Poisoning

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

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

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

TIC of lipophilic toxins spiked on a blank scallop sample (10 ugkg-1)

Chromatograph for PSP toxinsPSP screen: 14 min vs 40 min (HPLC) per sampleFull PSP confirmation: 56 min vs 160 min (HPLC) per sample

GTX5

Shellfish matrix

dcGTX2,3

dcGTX2,3GTX1,4

C3,4

C1,2

NEO

dcSTXdcNEO

dcSTXNEO

GTX2,3GTX1,4

STXNEO

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

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.

TIC of lipophilic toxins spiked on a blank OYSTER sample (10 ugkg-1)

Expanded +MRM of lipophilic spiked oyster sample

GYM

DASPX

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

Now

UPLCs coupled with MS/MS or Fluorescence detector

(Running time < 8 min per injection)

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

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

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

Biotoxin detections in Australia – past 2 years

DSPASPPSP

PSPASPDSP

DSPASPPSP

DSPASPPSP

DSPASPPSP

0

1000

2000

3000

4000

5000

6000

7000

8000

0

2

4

6

8

10

12

14

16

18

15-Oct-12 15-Jan-13 15-Apr-13 15-Jul-13 15-Oct-13 15-Jan-14 15-Apr-14 15-Jul-14

Algal cell concentration(cells per litre)

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

dcGTX2,3GTX1,4

High C3,4

GTX5

C1,2

Low GTX2,3

STX

C1,2

dcGTX2,3

GTX5 STX

Low GTX2,3

dcSTX or NEO

dcSTX, No NEO in POX Peroxide Oxidation

Periodate Oxidation

PSP in Gymnodium bloom

PSP in Alexandrium blooms

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0

1

2

3

4

5

6

7

15-Oct-12 15-Jan-13 15-Apr-13 15-Jul-13 15-Oct-13 15-Jan-14 15-Apr-14 15-Jul-14

Algal cell concentration(cells per litre)

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

dcGTX2,3High GTX1,4

No C3,4High

GTX2,3

High C1,2

Very low GTX5

STX

dcSTX or NEO

High GTX2,3

High C1,2

dcGTX2,3

Peroxide Oxidation

Periodate Oxidation

Fast accumulation and degradation of PSP in shellfish

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

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

Acknowledgement:

Tasmanian Shellfish Quality Assurance Program – Megan, Jason & Howel

Advanced Analytical Biotoxin team – Rama, Feng & Dave

Andrew BradburyAdvanced Analytical AustraliaPh: 07 3268 1228andrew.bradbury@advancedanalytical.com.au