LC QQQ MARINE BIOTOXINS Dr. Jerry Zweigenbaum Agilent Technologies Agilent Americas Food Group
Breaking News... Recently, legislation in the EU introduced liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) as the soon to be, reference method for the detection of lipophilic marine biotoxins in live bivalve molluscs, tunicates and echinoderms. Annex 1, SCoFCAH, 18-11-2010, SANCO/6831/2009 rev 7 amending EU Regulation 2074/2005. This change in legislation allows for significant developments in the area of mass spectrometric detection for phycotoxins.
Marine Biotoxins: Why analyse for them? Marine biotoxin related illness can range from headaches, vomiting and diarrhoea to neurological problems, and in extreme cases can lead to death.
Is it really an issue? Agilent FC 24 Website RASFF Alert notifications are sent when a food or feed presenting a serious health risk is on the market and when rapid action is required. The RASFF member that identifies the problem and takes the relevant actions (e.g. withdrawal of the product) triggers the alert and allows other countries to search there own outlets.
Responsibilities for Producers of shell fish They must ensure their product must not contain marine biotoxins in quantities that exceed... 800 micrograms per kilogram for paralytic shellfish poison (PSP), 20 milligrams of domoic acid per kilogram for amnesic shellfish poison (ASP) 160 micrograms of okadaic acid equivalents per kilogram for okadaic acid, dinophysistoxins and pectenotoxins in combination, 1 milligram of yessotoxin equivalents per kilogram for yessotoxins, 160 micrograms of azaspiracid equivalents per kilogram for azaspiracids.
Site Closures 2005 Ireland (locality) The picture before harvesting Assessment of Irish beds in 2005 Yellow: ASP Red: DSP Blue: AZP Green: DSP+AZP Courtesy of Dr. Philipp Hess, Ifremer
Examples of shell fish poisoning incidents Examples of shellfish poisoning incidents. Note that no new toxin groups have been reported since the discovery of azaspiracids in 1995 (Hess et al., 2008). Large-scale poisoning events for okadaic acid group toxins have still occurred during the last decade despite the toxic algae and toxins involved being known for over 20 years. POISONING DSP 164 NO. OF CASES DSP > 300 ASP 107 PSP 187 NSP 48 AZP 24 DSP > 300 DSP 200 DSP 159 SHELLFISH LOCATION SPECIES OF ILLNESS REFERENCE MUSSELS AND SCALLOPS JAPAN YASUMOTO ET AL., 1978 BLUE MUSSELS (M. EDULIS) NORWAY, SWEDEN UNDERDAHL ET AL., 1985 BLUE MUSSELS (M. EDULIS) CANADA PERL ET AL., 1990 CLAMS (A. KINDERMANII) GUATEMALA RODRIQUE ET AL., 1990 EASTERN OYSTER (C. VIRGINICA) UNITED STATES MORRIS ET AL., 1991 BLUE MUSSELS (M. EDULIS) IRELAND MCMAHON AND SILKE, 1998 BLUE MUSSELS (M. EDULIS) BELGIUM DE SCHRIJVER ET AL., 2002 BROWN CRAB (C. PAGURUS) NORWAY AUNE ET AL., 2006 BLUE MUSSELS (M. EDULIS) UNITED KINGDOM UK COT, 2006 Provided courtesy of Dr. Philipp Hess, Ifremer
During the last 2 years EFSA publish scientific opinion that bioassay has shortcomings and is not considered an appropriate tool for control of some toxins in Shellfish because of the high variability in results, the insufficient detection capability and the limited specificity...a key, dirty dozen is the key focus. Ring trials take place across Europe. As a result an LCMSMS approach becomes effectively validated by the Union Reference Laboratory for marine biotoxins and is declared to be an approach which maintains and ensures a full protection of consumer health without the shortcomings of the biological test, such as the high variability in results, the insufficient detection capability and the limited specificity. In November Member States endorsed the relevant European Commission proal during meeting of the Standing Committee of the Food Chain and Animal Health (SCoFCAH).
In Annex III to Regulation (EC) No 2074/2005, Chapter III to be replaced by... LIPOPHILIC TOXIN DETECTION METHODS: Chemical methodology The EU-RL LC-MS/MS method shall be the reference method for the detection of marine toxins as referred to in Chapter V(2)(c), (d) and (e) of Section VII of Annex III, to Regulation (EC) No 853/2004. This method shall determine at least the following compounds: - okadaic acid group toxins : OA, DTX1, DTX2, including their esters - pectenotoxins group toxins : PTX1 and PTX2, - yessotoxins gp toxins: YTX, 45 OH YTX, homo YTX, and 45 OH homo YTX, -azaspiracids group toxins: AZA1, AZA2 and AZA3. Total toxicity equivalence shall be calculated using toxicity equivalent factors (TEFs) as recommended by EFSA. If new analogues of public health significance are discovered, they should be included in the analysis. Total toxicity equivalence shall be calculated using toxicity equivalent factors (TEFs) as recommended by EFSA.
In Annex III to Regulation (EC) No 2074/2005, Chapter III to be replaced by... LIPOPHILIC TOXIN DETECTION METHODS: Biological methods A series of mouse bioassay procedures, may be still used until 31 December 2014 for detecting marine toxins. After that period, the mouse bioassay shall be used only during the periodic monitoring of production areas and relaying areas for detecting new or unknown marine toxins on the basis of the national control programmes elaborated by the Member States.
Dirty Dozen lipophilic toxins: Compound Compound class Toxicity factor OA DSP 1 DTX1 DSP 1 DTX2 DSP 0.6 AZA1 AZP 1 AZA2 AZP 1.8 AZA3 AZP 1.4 Compound Compound class Toxicity factor PTX1 DSP 1 PTX2 DSP 1 YTX DSP 1 homo-ytx DSP 1 OH-YTX DSP 1 OH-homo-YTX DSP 0.5
Determination of lipophilic marine biotoxins in shellfish using Triple Quadrupole LC/MS/MS Experimental Work and Data provided by: Oliver Keuth Chemical and Veterinary Analytical Institute Münsterland-Emscher-Lippe, Münster, Germany Dr. Begoña Ben Gigirey, Prof. Ana Gago-Martinez European Union Reference Labatory for Marine Biotoxins (EU-RL-MB) and Dept. Analytical and Food Chemistry, Faculty of Chemistry, University of Vigo
Review of lipophilic marine biotoxin analysis: There are 2 LC-MS/MS methods proed for the analysis of lipophilic marine biotoxins and both methods are currently evaluated in ring trials: Acidic method (based on McNabb et al., 2005) Reverse phase conditions Acidic mobile phases Electrospray ionization with polarity switching Multiple reaction monitoring Alkaline method (based on Gerssen et al., 2009) Reverse phase conditions Basic mobile phases (ph 11) Electrospray ionization with negative and itive time segment Multiple reaction monitoring
Influence of ph value of mobile phase: DTX-1: O O C H 3 CH 3 H H O HO H O H OH CH 2 O H C H 3 CH 3 HO O O H C H 3 OH OH O +H + O O C H 3 CH 3 H H O HO H O H OH CH 2 O H C H 3 CH 3 HO O O H O - H 3 C OH O Gerssen et al., J. Chromatogr. A, 1216 (2009) 1421-1430
Reaction Monitoring Quad Mass Filter (Q1) Quad Mass Filter (Q3) Collision Cell Spectrum with background ions (from ESI) 210 222 Q1 lets only target ion 210 pass through 210 Collision cell breaks ion 210 apart Q3 monitors only characteristic fragments 158 from ion 210 for quant 165 268 280 158 191 210 158 170 210 250 290 190 210 150 170 190 210 160 Chromatogram High background Low background
Ionization of lipophilic toxins in electrospray Compound class Formula ESI mode Precursor ion Okadaic acid group OA DTX1 DTX2 Azaspiracids AZA1 AZA2 AZA3 Pectenotoxins PTX1 PTX2 Yessotoxin group YTX homo-ytx OH-YTX OH-homo-YTX C 44 H 68 O 13 C 45 H 70 O 13 C 44 H 68 O 13 C 47 H 71 NO 12 C 48 H 73 NO 12 C 46 H 69 NO 12 C 47 H 70 O 15 C 47 H 70 O 14 C 55 H 82 O 21 S 2 C 56 H 84 O 21 S 2 C 55 H 82 O 22 S 2 C 56 H 84 O 22 S 2 negative itive negative itive negative itive itive itive itive itive itive itive itive negative negative negative negative negative negative negative negative [M-H] - = 803.5 [M+Na] + = 827.5 [M-H] - = 817.6 [M+Na] + = 841.5 [M-H] - = 803.5 [M+Na] + = 827.5 [M+H] + = 842.6 [M+H] + = 856.5 [M+H] + = 828.5 [M+NH 4 ] + = 892.5 [M+Na] + = 897.5 [M+NH 4 ] + = 876.6 [M+Na] + = 881.5 [M-H] - = 1141.5 [M-2H] 2- = 570.3 [M-H] - = 1155.4 [M-2H] 2- = 577.4 [M-H] - = 1157.4 [M-2H] 2- = 578.4 [M-H] - = 1171.4 [M-2H] 2- = 585.4 AZA1 PTX2 YTX
Collisionally induced dissociation of OA itive CE 45 V negative CE 60 V
Application Number 1: Acidic Method Single polarity
Methodology Sample preparation: 2 g cooked and grinded mussel tissue Extraction with aqueous solvent (80 % methanol) Shaking or blending mixture and centrifugation Repeat extraction, supernatants decanted into volumetric flask, filled up to 50 ml Filter extract, using 0.45 µm membrane filter Inject 10 µl LC-MS/MS
Methodology HPLC conditions: Column: Flow rate: C-18, 150 x 2 mm, 5 µm (itive mode) Agilent ZORBAX Eclipse Plus C-8, 75 x 4.6 mm, 3.5 µm (negative mode) 0.2 ml/min Column temp: 30 C Injection volume: 10 μl (needle wash in flushport 5 seconds) Solvent Channel A: Solvent Channel B: 0.1% formic acid (itive mode) 2 mm ammonium acetate (negative mode) methanol Gradient: Time (min) A (%) B (%) 0.0 90 10 10.0 10 90 22.0 10 90 23.0 90 10 30.0 90 10 Overall Cycle Time: 30.0 min
Methodology Agilent Jet Stream conditions: Drying gas temp: 300 C Gas flow rate: 5 L/min Nebulizer pressure: 45 psi Sheath gas temp: 250 C Sheath gas flow: 11 L/min Nozzle Voltage: +/- 500 V LC flow Super-heated N 2 sheath gas Nebulizer N 2 gas (near sonic velocity) delta EMV: Capillary voltage: 400 V +/- 3500 V Thermal focusing Electrospray
Methodology MRM transition parameters: Analyte Polarity Prec Ion m/z OA and DTX2 DTX1 PTX1 PTX2 PTX2sa * YTX neg neg 827.5 827.5 841.5 841.5 897.5 897.5 881.5 881.5 899.5 899.5 1141.5 1141.5 Prod Ion m/z 723.4 809.2 737.2 823.2 555.3 853.5 539.3 837.5 855.5 557.3 1061.3 925.5 Frag [V] 220 220 220 220 230 230 230 230 230 230 CE [ev] 55 45 55 45 70 60 70 60 60 70 Quantifier Homo-YTX * neg 1155.4 1075.5 135 35 X OH-YTX neg 1157.4 1077.5 135 35 X OH-Homo-YTX * neg 1171.4 1091.5 135 35 X AZA1 AZA2 AZA3 842.5 842.5 856.5 856.5 828.5 828.5 824.5 806.5 838.5 820.5 810.5 792.5 135 135 200 200 200 200 200 200 35 60 40 55 40 55 40 X 55 * Transitions based on literature information X X X X X X X X
Chromatograms for blue mussel extract and QC Blue mussel extract AZA1 to AZA3: < 20 µg/kg OA: 37 μg/kg DTX2: 120 μg/kg DTX1: 69 μg/kg QC-sample AZA1: OA: PTX2: 15 µg/kg 15 μg/kg 15 μg/kg
Lipophilic marine toxins calibration curves Calibration curve (matrix matched) AZA1, OA, PTX2, and YTX reference standards spiked into blue mussel extract Calibration range: AZA1: OA: 2.5 to 25 ng/ml 1 to 38 ng/ml
Quantitation of marine toxins There are currently just 4 reference compounds (AZA1, OA, PTX2, YTX) available from the National Research Council, Institute for Marine Biosciences (NRC-CNRC), Halifax, Canada All other compounds need to be quantified based on the calibration response of related compounds. OA reference standard (10 ng/ml) Blue mussel extract containing OA and DTX-2 (isomeric compounds)
Quantitation of DTX2 based on OA calibration Area of OA for each calibration point is copied to DTX2 and allows automated quantitation of DTX2 based on calibration of OA Other to application of average response factors, concept is amenable to standard addition, matrix spikes, QCs etc. Baseline separation of signals is mandatory if compounds are isomeric; too good separation can have effect on quantitation result
Method validation The method has been validated within an international collaborative study. The collaborative study was conducted in the framework of the working group 64 LFGB Phycotoxins, which is hosted by the federal Office of Consumer Protection and Food Safety (BVL). LODs and LOQs in cooked, grinded mussels: Compound LOD LOQ 1 OA 6 μg/kg 20 μg/kg 1 DTX1 & 2 6 μg/kg 20 μg/kg 2 AZA1 to 3 6 μg/kg 20 μg/kg 1 PTX1 & 2 6 μg/kg 20 μg/kg 3 YTX 10 μg/kg 35 μg/kg 1 MRL in raw mussel material for sum of OA, DTX1 & 2, PTX1 & 2: 160 μg/kg OA equivalents 2 MRL in raw mussel material for sum of azaspiracids: 160 μg/kg AZA1 equivalents 3 MRL in raw mussel material for sum of yessotoxins: 1000 μg/kg YTX equivalents
OA DTX2 DTX1 AZA3 PTX2 45 OH-YTX/ 45 OH-homo-YTX YTX/Homo-YTX AZA2 AZA1 Application Number 2: Alkaline Method, both polarities Based on a method published by Gerssen et al., 2009 Alkaline mobile phase @ ph 11 Chromatographic separation of compounds ionized in itive and negative mode Negative ionization Positive ionization Peak shapes of azaspirazids negatively effected Data provided by courtesy of Prof. Dr. Ana Gago-Martinez, EURLMB, Vigo
Methodology HPLC conditions: Column: Flow rate: Waters X-Bridge C18, 150 3 mm, 3.5 μm 0.4 ml/min Column temp: 40 C Injection volume: 10 μl (needle wash in flushport 5 seconds) Solvent Channel A: 0.05% ammonia (v/v) in water (ph 11) Solvent Channel B: 0.05% ammonia (v/v) in 90% acetonitrile Gradient: Time (min) A (%) B (%) 0.0 90 10 1.0 90 10 10.0 10 90 13.0 10 90 15.0 90 10 Overall Cycle Time: 20.0 min
Methodology MRM transition parameters: Analyte Polarity Prec Ion m/z Prod Ion m/z Frag [V] CE [ev] Quantifier OA and DTX2 neg neg 803.5 803.5 255.2 113.1 330 330 52 66 X DTX1 neg neg 817.6 817.6 255.2 113.1 330 330 52 66 X PTX1 892.5 892.5 839.5 213.1 170 170 22 42 X PTX2 876.6 876.6 823.7 213.1 170 170 22 42 X YTX neg neg 570.3 570.3 467.1 396.2 185 185 30 34 X Homo-YTX neg 577.4 577.4 474.4 403.3 185 185 30 34 X OH-YTX neg 578.4 578.4 467.3 396.3 185 185 30 34 X OH-Homo-YTX neg 585.4 585.4 474.4 403.3 185 185 30 34 X AZA1 842.6 842.6 824.6 672.4 220 220 29 53 X AZA2 856.5 856.5 838.5 672.4 220 220 29 53 X AZA3 828.5 828.5 810.5 658.4 220 220 29 53 X
OA PTX2 YTX AZA1 OA DTX2 DTX1 AZA3 PTX2 AZA2 45 OH-YTX YTX AZA1 Chromatograms for mussel extract and QC Mussel extract 45-OH-YTX: 214 µg/kg OA: 133 μg/kg DTX2: 125 μg/kg YTX: 380 µg/kg DTX1: 31 μg/kg AZA3 25 µg/kg AZA1 81 µg/kg AZA2: 18 µg/kg PTX2: 5 µg/kg QC-sample OA: 80 μg/kg (100.3%) YTX: 500 µg/kg (102.7%) AZA1: 80 µg/kg (102.6%) PTX2: 80 μg/kg (99.5 %) Data provided by courtesy of Prof. Dr. Ana Gago-Martinez, EURLMB, Vigo
Lipophilic marine toxins calibration curves Calibration curve (matrix matched) AZA1, OA, PTX2, and YTX reference standards spiked into mussel extract Calibration range: AZA1, OA and PTX2: YTX: 2 to 24 ng/ml 12.5 to 150 ng/ml AZA1 Data provided by courtesy of Prof. Dr. Ana Gago-Martinez, EURLMB, Vigo YTX
Quality control criteria Chromatographic resolution OA/DTX 1.0 Sensitivity based on S/N of qualifier ion: S/N 3 for lowest standard (matrix matched) Linear calibration curves with a correlation coefficient r 2 0.98 for at least five calibration points Response drift over sample series 25% measured via the slope variation of the two sets of calibration curves Carry-over less than 10% of lowest calibration point Retention time drift less than 3% ** EURLMB results from pre-trial
DTX-2 DA GYM SPX1 45 OH-YTX/ 45 OH-homoYTX YTX/ homoytx OA DTX1 PTX2 AZA3 AZA1 AZA2 Where to go from here? Add more compounds where applicable Improve chromatographic separation and reduce runtimes by using UHPLC (requires intelligent management of duty cycles) Make use of fast polarity switching capabilities of the G6460A QQQ system for extra speed Reduce injection volumes for enhanced robustness * Real mussel extract, responses normalized to 100%
Methodology HPLC conditions: Column: ZORBAX RRHD SB C-8, 50 x 2.1 mm, 1.8 µm Flow rate: 0.4 ml/min Column temp: 40 C Injection volume: 5 μl (needle wash in flushport 5 seconds) Solvent Channel A: Solvent Channel B: 2mM ammonium formate + 25 mm formic acid in water 2mM ammonium formate in 95% acetonitrile Gradient: Time (min) A (%) B (%) 0.0 88 12 0.5 88 12 3.0 50 50 6.5 10 90 7.0 10 90 7.1 88 12 Overall Cycle Time: 8.0 min
YTX OA DTX1 PTX2 DA AZA1 GYM SPX1 DA GYM SPX1 45 OH-YTX/45 OH-homoYTX YTX/homoYTX OA DTX-2 DTX1 PTX2 AZA3 AZA2 AZA1 Chromatograms for mussel extract and QC Mussel extract DA: 1490 μg/kg GYM: 7 μg/kg SPX1: 3 μg/kg OA: 720 μg/kg DTX2: 60 μg/kg YTX: 230 µg/kg DTX1: 100 μg/kg AZA3 25 µg/kg AZA1 150 µg/kg AZA2: 4 µg/kg PTX2: 7 µg/kg QC-sample DA: 787 μg/kg (109.9%) GYM: 27 μg/kg (111.1%) SPX1: 30 μg/kg (109.8%) YTX: 38 µg/kg (134.6%) OA: 31 μg/kg (107.7%) PTX2: 25 μg/kg (105.3%) DTX1: 4 μg/kg (110.6%) AZA1: 10 µg/kg (105.5%)
Lipophilic marine toxins calibration curves Calibration curve (matrix matched) DA, GYM, SPX, OA, DTX1, AZA1, OA, PTX2, and YTX reference standards spiked into mussel extract Calibration range: AZA1: OA: PTX2: YTX: 0.1 to 46 ng/ml 0.2 to 200 ng/ml 0.2 to 120 ng/ml 0.3 to 180 ng/ml AZA1 Solid points: Blue triangles: Calibration before sample series Calibration at end of sample series YTX
New triggered MRM functionality for confirmation Improved identification by triggering additional confirmatory MRMs if primary MRMs are detected above threshold Triggered MRM advantageous over data dependant product ion scan because: more sensitive Peak shapes of primary transitions are not compromised (constant cycle time) Stable in-spectra fragment ratios User defined library with search capabilities in MassHunter software. Page 39
Identification of compounds by library search Library search functionality in MassHunter Qual Excellent peak shapes for primary transitions! Page 40
Identification of compounds in batch review Page 41
Summary and Outlook There are two methods for lipophilic shellfish toxins currently evaluated in international ring trials (alkaline method vs. acidic method) We have methods for both approaches available and our system has proved its sensitivity and robustness in both ring trials There is a special script for the MassHunter Quant software available to deal with the lack of reference compounds An application note together with CVUA Münster is now available for the analysis of lipophilic toxins under acidic conditions (5990-6377EN) A similar note will be published for the alkaline method in January together with the EURL for marine toxins in Vigo, Spain, followed by a note on an UHPLC method with /neg switching for extra speed Future work will focus on emerging toxins like Palytoxin, Brevetoxins, Ciguatoxins by LC-QQQ and the use of accurate mass instruments for the identification of toxins and toxin metabolites.
Thank you for you attention Questions about the application note, please contact: Dr. Thomas Glauner EMEA LC/MS Food Segment Scientist Chemical Analysis Group Agilent Technologies Sales & Services GmbH & Co.KG Hewlett-Packard-Str. 8 76337 Waldbronn Phone: +49 (0)7243 602-2719 thomas_glauner@agilent.com