Introduction quantitation of molecular ions would lead to the

Introduction

Identification and characterization of ciguatoxin implicated in reef fishes along Indian EEZ become a new challenge to fisheries sector in terms of food and consumer safety. Due to the complex structure and existence of different congeners present in individual toxin, bioassay directed physico-chemical methods are used for the determination of these toxins (Holland, 2008). Bioassays depend on the response of tested animals or cultures cells would aid in the assessment of toxicity and related effects. Sometimes these assays are not useful as a market test for individual toxins and only remains as a toxicological tool for many laboratories and ethical concerns over animal study also make difficulties in the regular monitoring of food safety issues related with toxins. In this context, Physico-chemical methods based on chromatographic and spectroscopic techniques play a vital role in the quantitative analysis of known seafood toxins. Different instrumentation methods are applicable in the field of marine algal toxin research viz., Gas Chromatography, thin-layer chromatography (TLC), HPLC-UVD/FLD, LC-MS, and capillary electrophoresis (CE) etc., in which mass spectrometry with Electron Spray Ionization (ESI) or Atmospheric pressure Ionization mode (API) and Multiple Reaction Monitoring (MRM) transitions would help in the identification of molecular ions of individual toxins and its congeners in a mixture or complex form of compound. Tandem Mass Spectrometry allows universal detection capability, high sensitivity, selectivity and specificity in detection of complex molecules. Through the precise and accurate quantitation of molecular ions would lead to the identification of novel toxins from a complex mixture and routine monitoring of food safety.

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LC-MS/MS is a combination of LC system for separation of analyte, an ionization interface to ionize the molecules, and a MS in which the ions are separated according to their mass-to-charge ratio (m/z) and detected in a high vacuum environment (McMaster, 2005). ESI, APCI, APPI, FAB and MALDI are the key ionization interfaces used for detection and separation of analyte, in which ESI is the most common ionization interface for more polar compounds and it acquires both positive and negative ion spectra (Quilliam, 2003b, McNabb et al., 2005). The mass analyzer known as the heart of MS are of three types viz., quadrupole, ion trap (IT), and time of flight (TOF). MALDI-TOF-MS has been mostly used in the proteomic analysis or in the analysis of large biomolecules (Cai et al., 2005; McMaster, 2005; Moyer et al., 2002; Reyzer & Caprioli, 2007) and it cannot be combined with LC and provides relatively poor quantitative reproducibility (Cai et al., 2005). In ITMS relatively low dynamic range due to ion accumulation uses for quantitation and mass range is lower than TOF MS (Aebersold & Mann, 2003; March, 1997). TOF-MS can also obtain high mass resolutions to enable accurate mass measurements and provide the chemical formulae of novel toxins (Moyer et al., 2002). Triple quadrupole or QTRAP MS comprises Source (Q0), Q1 &Q3, collision cell (Q2) and Detector (internal structure given in Chapter 2). Q TRAP Mass Spectrometry in combination with ESI Ionization mode and MRM transitions are widely used in marine and fresh water algal toxin detection and delivers molecular mass and structural information in full scan and/or daughter ion scan mode, hence it is considered as a highly sensitive and selective detector for quantitative analysis of complex mixtures.

Ion Spray mass detection was first introduced by Lewis et al., 1994 and a CTX Rapid Extraction Method known as CREM was also developed by Lewis et al., 2009  There are 20 different congeners were identified for CTX from the Pacific, the Caribbean and the Indian Ocean regions (Caillaud et al., 2010). Potent polyether ciguatoxin responsible for foodborne disease accumulates the toxin at risk levels above 0.1ppb (Lewis et al., 2009).  CTX contain multiple classes of charged ions which are difficult to monitor by bioassays. In this study, Liquid Chromatography-Tandem Mass Spectrometry methods were used to compare novel compounds produced by ciguatoxin implicated reef fish L. bohar along Indian Coast. Two validated extraction method was followed for the isolation toxic eluents and compared the extracted ion chromatograms obtained for the previous reports of CTX molecular mass range.

Table 1: Group of CTXs isolated from fish and micro algal origin

Origin

Examples of CTX

Molecular weigh

Source

Reference

Pacific
Type I & Type II
(P-CTX)
 

CTX (CTX1B, CTX-1)
CTX2A2 (CTX-2, 52-epi-54-deoxyCTX)
CTX2B2 (CTX-3, 54-deoxyCTX)
CTX4A
CTX4B (GTX-4B, Gt 4b)

1110.6
1094.5
1060.8
1022.8
1056.0

Both Carnivorous and Herbivorous fishes and  Dinoflagellate G. toxicus

Murata et al., 1990 Lewis et al., 1991 Lewis et al., 1993
Satake et al., 1993

Caribbean
(C-CTX)

CTX-1 & CTX-2

1140.7

Carnivorous fish

Lewis et al., 1998

Indian Ocean
(I-CTX)

CTX-1, CTX-2, CTX-3 &  CTX-4

1140.6
1157.6

Carnivorous fish

Hamilton et al., 2002

 

A mass range that covers all known ciguatoxin (CTXs) and Palytoxin (PbTxs) include within the range m/z 700 to 1400 Daltons (Hamilton et al., 2002). All CTXs identified to date are heat stable polyether toxins and P-CTX-1 remains the most potent ciguatoxin characterized (Lewis et al., 1991), often contributing approximately 90% of the total lethality of carnivorous ciguateric fish capture in the western Pacific Ocean (Lewis and Sellin,1992), and posing a health risk at levels ? 0.1 ppb (Lewis, 2001).

Ciguatera Fish Poisoning confirmed from Lutjanus bohar through Mouse Bioassay method. Working with trace quantities of CTX extract is always make the extraction methods and detection of CTX very difficult task.in this chapter an attempt was made in to the optimization of CTX using size exclusion chromatography and Solid Phase Extraction methods using High Performance Liquid Chromatography and Mass Spectrometry.