UPLC-MS/MS method for the routine quantification of regulated and non-regulated lipophilic marine biotoxins in shellfish

Transcription

1 UPLC-MS/MS method for the routine quantification of regulated and non-regulated lipophilic marine biotoxins in shellfish ArjenGerssen*, MirjamKlijnstra*, Antonietta Wallace and Simon Hird *Work was carried out in collaboration with RIKILT 215 Waters Corporation 1

2 Overview Introduction Methodology The emergence of the use of LC-MS/MS Objectives Move to UPLC-MS/MS Experimental design and conditions Results and discussion Conclusions and future work 215 Waters Corporation 2

3 Introduction Depending on climatic conditions, harmful algae blooms (HAB) can occur throughout the coastal regions of Europe These species produce marine biotoxins(mbtxs), which are ingested by filter feeding organisms, accumulating within their flesh During these bloom events, filter feeding bivalve molluscs can accumulate relatively high concentration levels of toxins Although MBTXs are not often toxic to these species, they may produce severe effects on humans if seafood contaated with MBTXs is consumed GerssenA et al. (21) Toxins 2, ; doi:1.339/toxins Waters Corporation 3

4 Introduction MBTXs can be classified according to polarity into two major groups: Hydrophilic toxins, such as domoicacid and saxitoxinscan produce amnesic shellfish poisoning or paralytic shellfish poisoning respectively Lipophilic toxins comprise several families of compounds: o Azaspiracids(AZAs) o Pectenotoxins(PTXs) o Spirolids(SPXs) o Yessotoxins(YTXs) o Brevetoxins(BTXs) o Ciguatoxins(CTXs) o Okadaic acid group (OA) including dinophysistoxin derivatives(dtxs) Their toxic effects are diverse including gastrointestinal disorders or neurological symptoms 215 Waters Corporation 4

5 Introduction In the European Union, Regulations(EC) No 853/24 and (EU) 786/213 establish the maximum permitted limits of lipophilic MBTXs in live bivalve molluscs and these toxins should be monitored in official control programs The legislated group of lipophilicmbtxs consists of four different chemical groups: Okadaic acid (OA) and its derivatives, dinophysistoxins(dtxs) Yessotoxins(YTXs) Azaspiracids(AZAs) Pectenotoxins(PTXs) These are the most frequently toxins that have appeared along European coasts, together with cyclic ies (CIs), such as the spirolides(spxs), for which no legislation has yet been established Only few analogues of each group are legislated, and their quantity has to be referred to a predoant compound of the group, called the reference compound 215 Waters Corporation 5

6 Structures of regulated (a) and nonregulated (b) lipophilic MBTXs a) b) Toxin R 1 R 2 Okadaicacid CH 3 H Dinophysistoxin-1 CH 3 CH 3 Dinophysistoxin-2 H CH 3 Toxin R 1 R 2 Azaspiracid-1 H CH 3 Azaspiracid-2 CH 3 CH 3 Azaspiracid-3 H H Toxin R 1 R 2 R 3 R 4 Pinnatoxin-E H OH CH 3 Pinnatoxin-F H OH CH 3 Toxin R 1 Pectenotoxin-1 OH Pectenotoxin-2 H Pinnatoxin-G OH H H Toxin R 1 n Yessotoxin H 1 Homo Yessotoxin H 2 45OH Yessotoxin OH 1 45OH Homo Yessotoxin OH 2 13-desmethyl spirolide C Gymnodie 215 Waters Corporation 6

7 Monitoring for official control Regulation (EC) 854/24 requires the Competent Authority for food safety to establish an monitoring programme of classified shellfish relaying and production areas to check for the possible presence of toxin producing phytoplankton in the water and MBTXs in the shellfish flesh Shellfish samples, relevant for each location, are tested for MBTXs for which maximum permitted levels are set by the EU legislation, including lipophilic MBTXs Major species of commercial interest in the UK are common mussels (Mytilisedulis), common cockles (Cerastodermaedule), Pacific oyster (Crassostrea gigas), native oyster (Ostrea edulis), king scallops (Pecten maximus), Queen Scallops (Aequipectenopercularis), hard clams (Mercenaria mercenaria), and razor clams (Ensis spp.) Monitoring frequency for flesh testing is based on a risk assessment e.g.in Scotland; weekly in March to December and monthly in January and February 215 Waters Corporation 7

8 Other monitoring All monitoring results in the UK are published weekly on the shellfish monitoring page of the FSA (for England, Wales and Northern Ireland) and FSAS (Scotland) websites The results from such monitoring for official control are used to inform decisions taken by Food Business Operators regarding harvesting activity and the need to increase the amount of end product testing (EPT) that may be required to demonstrate product safety and reduce the risk of toxic shellfish being placed on the market When legal limits of MBTXs in shellfish are breached, the competent authority and local authorities take action to ensure the affected areas are closed for harvesting Reporting presence of MBTX at lower concentrations provides opportunities for trigger points for action by competent authorities, harvesters or food businesses themselves 215 Waters Corporation 8

9 The EU maximum permitted levels of lipophilic MBTX in shellfish Toxin group Okadaicacid (OA), dinophysistoxins (DTX) and pectenotoxins (PTX) Yessotoxins(YTX) Maximum permitted level (MPL) Amber flesh trigger level 16µg of okadaic acid 8 µg/kg equivalents/kg 3.75mg yessotoxin equivalent/kg Red flesh trigger level 16 µg/kg 1.8 mg/kg 3.75mg/kg Azaspiracids (AZA) 16µg azaspiracid equivalents/kg 8 µg/kg 16 µg/kg 215 Waters Corporation 9

10 Original methodology Mouse bioassay was the reference method for the official control of lipophilic MBTXs for many years This assay provides information about total toxicity, but has several drawbacks; Ethical issues Lack of selectivity: matrix interference Limited sensitivity for some compounds (e.g. OA group) Poor reproducibility of results From July 214, in Europe, it was no longer possible officially to analyze for the presence of lipophilicmbtxs in shellfish by MBA (EU Commission Reg 15/211, 211) There are screening tests available suitable for EPT for DSP only Antibody based tests (lateral flow or ELISA) Phosphatase Inhibition Assay (PP2A) 215 Waters Corporation 1

11 A change in methodology LC-MS/MS methods were developed as alternatives In 211, a method based upon LC-MS/MS was established as the reference method for the detection of lipophilicmbtxs and used as matter of routine, both for the purposes of official controls at any stage of the food chain and EPT by food business operators EU-Harmonised Standard Operating Procedure for deteration of Lipophilicmarine biotoxinsin molluscs by LC-MS/MS Version 5, January 215 The EU reference method is based on: A fixed extraction procedure Separation using RP LC using either an acidic mobile phase or alkaline mobile phase Detection by tandem quadrupole MS 215 Waters Corporation 11

12 LC-MS/MS methodology A major advantage of using basic LC conditions is that toxins can be clustered in retention time windows separated for positively and negatively ionised molecular ions [1] The method was accessible to instruments unable to conduct rapid polarity switching [2] The method was originally validated for deteration in crude methanolicshellfish extracts and for extracts purified with [3] and without SPE [4] using LC-MS/MS 1. Gerssen A et al. (29) J. Chromatogr. A 1216: Rubies A et al. (215) J. Chromatogr. A 1386: Gerssen A et al. (21) Anal. Bioanal. Chem. 397: van den Top H et al. (211) Food Addit. Conta. 28: Waters Corporation 12

13 Project Aims Produce a faster method using alkaline conditions HPLC = 15 s Expected turnaround times are often 3 days from sample collection to report... Develop the method for regulated and some non-regulated MBTXs: cyclic ie compounds Test method on different matrices Obtain single day lab validation data 215 Waters Corporation 13

14 Experimental design The method was validated using EU Commission Decision 22/657/EC as a guideline Seven replicates, at each of the three spiking levels (.5, 1 and 1.5 times the MPL), were analysed Analysis was carried out on separate occasions using different types of blank shellfish extract 4 mussels (Mytilus edulis), 1 oysters (Crassosrea gigas), 1 cockles (Cerastoderma edule) and 1 ensis (Ensis directus) The accuracy, intraday precision (intraday repeatability, RSD r ), interdayprecision (within-laboratory reproducibility, RSD R ), linearity, decision limit (CCα), specificity and ruggedness were detered for the MBTXs for which reference standards were available Calibration was by matrix-matched calibration to mitigate matrix effects 215 Waters Corporation 14

15 Sample preparation and extraction The shellfish meat was homogenized with an Ultra Turrax Homogenized whole flesh shellfish tissue (1 g) was extracted with methanol Extract was vortex-mixed and centrifuged Procedure repeat (x3 total) Supernatant was transferred to a 1 mlvolumetric flask and made up to 1 mlwith methanol Crude shellfish extracts were filtered prior to analysis In order to detere the amount of DTX3 (ester forms of OA, DTX1 and -2) extracts were also subjected to alkaline hydrolysis using 2.5 M sodium hydroxide After heating the alkaline mixture for 4 at 76 C, the mixture was cooled to room temperature and subsequently neutralised using 2.5 M hydrochloric acid 215 Waters Corporation 15

16 LC-MS/MS conditions ACQUITY UPLC with FL autosampler Runtime: 5 Column: ACQUITYUPLC BEH C µm, mm Column temp: 4 C Mobile phase A: 6.7 mmnh 4 OH(aq.) Mobile phase B: 9:1 ACN:H 2O mmnh 4 OH Flow rate:.6 ml. -1 Injection volume: 5 µl Gradient: Time () A B Xevo TQ-S Ionisation mode: ESI Polarity: AZA- and PTX2-groups; positive OA- and -YTX-groups; negative Capillary voltage: 3. kv Source temperature: 15 C Desolvation temperature: 5 C Desolvationgas:8l.hr -1 MRM dwell time:.3 s Initial Waters Corporation 16

17 MS optimisation MS conditions were optimised by tuning (Intellistart) the MBTXs for which reference standards were available compounds Not all regulated MBTXs had standards available PTX1, 45OH YTX, homoytxand 45OH homoytx In order to detere these toxins, MRM transitions were included in the method based on the structural relation with the toxin from which a standard is available, PTX2 and YTX, respectively Furthermore, MRM transitions of other non-regulated lipophilic toxins were included to be used for screening For each compound two MRMs were optimised to allow the use of ion ratios for identification 215 Waters Corporation 17

18 MS/MS conditions Compound name trinor YTX YTX homoytx 45OH YTX 45OH Homo YTX COOH YTX COOH OH YTX COOH Homo YTX OAand DTX2 DTX1 Precursor (m/z) Product (m/z) ESI mode Cone (V) Collision (ev) Standard available No Yes No (Yes) No No No No No Yes Yes 215 Waters Corporation 18

19 MS/MS conditions Compound name Precursor (m/z) Product (m/z) ESI mode Cone (V) Collision (ev) Standard available AZA Yes AZA Yes AZA Yes AZA1 and Yes AZA No AZA No AZA4 and No AZA Yes PTX No PTX Yes PTX No PTX2sa No GYM Yes SPX Yes PinTX-G Yes 2-Me SPX G No PinTX-F Yes PinTX-E Yes 215 Waters Corporation 19

20 Results Chromatograms showing 2 MBTXs from the negative ion segment; blank and lowest matrix-matched standard (YTX 125 µg/kg and OA 2 µg/kg) TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 YTX TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 YTX TQS1_13DEC211_DSP2_ OA TQS1_13DEC211_DSP2_2 1 OA TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 OA Waters Corporation 2

21 Results Chromatograms showing 2 MBTXs from the positive ion segment; blank and lowest matrix-matched standard (PTX2 2 µg/kg and PinF 25 µg/kg) TQS1_13DEC211_DSP2_ PTX TQS1_13DEC211_DSP2_2 PTX2 1 TQS1_13DEC211_DSP2_ PTX TQS1_13DEC211_DSP2_2 PTX2 1 TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 PinF TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 PinF Waters Corporation 21

22 Results Chromatograms showing issues with peak shape for asazaspiracids; blank and lowest matrix-matched standard (2 µg/kg) TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 AZA3 AZA3 TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 AZA3 AZA3 TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 AZA3 1 AZA3 AZA3 TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 1 AZA1 TQS1_13DEC211_DSP2_ TQS1_13DEC211_DSP2_2 AZA Waters Corporation 22

23 Results Chromatograms showing main MBTXs spiked into mussels at 1x MPL = toxins currently regulated = toxins currently non-regulated 215 Waters Corporation 23

24 Calibration graphs For all toxins the linearity of the matrix matches standard calibration curve was excellent (R 2 >.99) for the concentration range used ( MPL) Compound name: OA Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 1. Residual Conc 8 Response Conc 215 Waters Corporation 24

25 Results of the single day validation Compound MPL conc. (µg/kg) Recovery () RSD r () RSD R () OA DTX DTX YTX AZA AZA AZA PTX GYM SPX PinTX-E PinTX-F PinTX-G Waters Corporation 25

26 Trueness and precision The recoveries obtained for the toxins were good and ranged from 91 to 18 The exception to this was PinTX-E where a recovery of 122 was obtained o PinTX-E tends to form methyl esters when stored in methanol o Might explain the higher than expected recovery as well as the poor repeatability when compared to the other analytes For most toxins the repeatability observed was good PinTX-E and SPX-1 were somewhat higher than expected 215 Waters Corporation 26

27 The decision limit (CCα) At what measured analyteconcentration are you 95 sure that the true concentration in a sample is > MPL? i.e.is not a false positive CCα= PL + t x SD R PL is the permitted level for the toxins in µg/kg t is 1.64 from a one-tailed t distribution with P=.5 (i.e.95) SD R is the standard deviation of the RSD R Compliant Non Compliant Result of measurement. MPL CCα Uncertainty associated with the measurement 215 Waters Corporation 27

28 Results of the single day validation Compound MPL conc. (µg/kg) Recovery () RSD r () RSD rl () CCα (µg/kg) OA DTX DTX YTX AZA AZA AZA PTX GYM SPX PinTX-E PinTX-F PinTX-G Waters Corporation 28

29 Surveillance data In 213 CEFAS analysed a total of 2787 samples from inshore locations in Scotland for lipophilicmbtxs using the LC-MS/MS reference method 326 inshore samples breached MPL In 9 of these cases, the LC-MS/MS method provided an early warning, detecting low toxin levels either one or two weeks prior to closure There were unprecedented concentrations of the OA/DTX/PTX group, primarily affecting mussel sites and in particular the Highlands & Shetland Isles o This event persisted throughout summer and autumn, and results above MPL (>16µg OA eq./kg) continued to be recorded at a number of sites in the Shetland Isles into December 213 Two distinct AZA events were recorded in 213, the first of which resulted in the detection of AZAs in 182 samples of which five recorded results above the MPL (>16µg AZA1 eq./kg) 215 Waters Corporation 29

30 Conclusions Blooms of algae responsible for production of lipophilicmbtxs occur quite frequently within European waters Methods based upon UPLC-MS/MS are now available for the detection of relevant lipophilic marine toxins in shellfish These methods can help to provide rapid analysis to manage shellfish harvesting areas and to reduce the risk of intoxication Due to changes in climate and algae transport through ballast water future blooms and toxic episodes from so-called emerging MBTXs cannot be excluded Therefore, method development should continue to extend the scope of the methodology The European Union Reference Laboratory for Marine Biotoxins (EURLMB) is planning a validation exercise in 215 to extend the LC- MS/MS method to include DomoicAcid, Cyclic Ies and Brevetoxins 215 Waters Corporation 3

31 Future work The main reported drawback of the LC-MS/MS method when used to protect human health is that it can only be used to detere a limited number of toxins Target toxins are selected before the acquisition Misses any emerging MBTXs that are present Is non-targeted analysis with LC-HRMS(/MS) a viable alternative? There is a need for screening to trigger points rather than qualitative (yes/no) answers for practical shellfish management Commercial availability of MBTX reference standards is limited To be able to detere the toxicity of a seafood sample by LC- MS/MS one needs knowledge of the toxicity of each analogue present in a sample but also the concentration of the same analogue in order to translate analytical data into total toxicity 215 Waters Corporation 31