g.IV). - No permitidos

Internal standards are frequently used to improve the precision and accuracy of quantitative determination of target compounds by HPLC and LC-MS and related methods (Skoog et al., 2007). Ideally an internal standard is added at known concentration to a solution of every sample and blank prior to extraction. After analysis of sample extracts, any variations observed in the concentrations of the internal standard

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in the samples compared to that of the blank will ideally reflect variations also in the target compound. These variations can be accounted for by adjusting the target compound concentration by a correction factor or percentage. This is done to correct for random and systematic errors which can occur during the preparation of samples, chromatography, and detection using mass spectrometry. The internal standard used needs to be chemically and physically analogous to the analyte, providing a response that is similar to that of the target compound but which is sufficiently different that the two compounds are readily distinguishable (Skoog et al., 2007). Thus the response factor of the internal factor compared to that of the target compound is important. Internal standards can be those compounds that are structurally related, structurally similar or isotopically labeled analogues of the analyte (Skoog et al., 2007).

Relatively few of the chromatographic methods used to analyse DA in either seawater or shellfish samples have incorporated the use of an internal standard. In one study the reason given for this was the lack of commercially available isotopically labelled compounds appropriate for use (De la Iglesia et al., 2008). One of the first and most commonly used internal standards reported in the literature is dihydrokainic acid (DHKA: Fig. 2.1) (Pocklington et al., 1990; Besiktepe et al., 2008; Litaker et al., 2008; Bargu et al., 2011). DHKA was first used in combination with FMOC derivatised DA and HPLC with florescence detection (FD) (Pocklington et al., 1990). As a result of this other studies using this method have also chosen to use this internal standard. DHKA was primarily selected because it had a different retention time to DA, it was commercially available, and it is not known to exist naturally in marine samples.

Another internal standard which has occasionally been used is kainic acid (KA: Fig. 2.1) which has been applied to HPLC-FD (FMOC) (Maroulis et al., 2008) and to one

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LC-MS method (Lawrence et al., 1994). To the knowledge of the present author, KA is the only internal standard reported to be used for LC-MS DA determination and only one study reports its use (Lawrence et al., 1994). Even the latter was unknown to the author at the beginning of the present study. Lawrence et al., (1994) found the compound to be effective in providing more reliable quantitative results for DA in shellfish tissues. One of the prime reasons given for its use as an internal standard was its availability, as the preferred option of isotopically labelled DA was not commercially availability. Lawrence et al., (1994) incorporated KA into their method to compensate for variations in the splitting ratio of LC effluents which was intended to reduce the flow rate of effluents reaching the electrospray interface. Lawrence et al., (1994) found that KA and DA peaks were clearly distinguishable due to their retention times. However, the retention times of both compounds appear to shift according to the type of sample analysed e.g. contaminated razor clams, crab meat, spiked rat urine, or spiked rat serum samples. It is not known whether these shifts are consistent within each sample type or whether they are a result of matrix effects and highly variable. There is no mention in works by Lawrence et al., (1994) as to the reproducibility of using KA as an internal standard and whether the target compound DA affects it. Furthermore, Lawrence et al., (1994) applied the use of KA as an internal standard for quantitative analysis of DA in seafood and biological samples, the application of this method for the analysis of DA in seawater and diatom samples has not been determined. KA has a relative molecular weight of 213 [protonated molecular ion m/z 214, M+H]+ and is structurally very similar, yet distinct, from DA (Fig. 2.1). The use of KA as an internal standard along with LC-MS methods for DA determination will be further explored in this chapter.

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Figure 2.1: Chemical structure of (A) domoic acid, (B) kainic acid, and (C) dihydrokainic acid.

C

B

A

Methoda SPE clean-upb Derivatisation reagent LOD

phytoplankton LOD shellfish tissue References

Mouse Bioassay 40 μg g-1 Quilliam, 2003

HPLC + UV DAD SAX-SPE 10-80 ng mL-1 0.5 μg g-1 Quilliam, 2003

HPLC + FD (FMOC) SAX-SPE

(shellfish only) 9-fluorenylmethylchloroformate 15 pg mL

-1 _{0.02-0.03 μg g}-1 Pocklington et al.,

1990

HPLC + FD (AQC) 6-aminoquinolyl-N-hydroxysuccinimidyl

carbonate 50 pg mL

-1 _{Sun and Wong, 1999}

HPLC + FD (NBD-F) 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole 1000 pg mL-1 0.006 μg g-1 James et al., 2000

HPLC + FD (NBD-CL) 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole 0.037 μg g-1 Maroulis et al., 2008

LC-MSn SAX-SPE 0.025 μg g -1 (MS1) 0.008 μg g-1 _(MS3₎ Furey et al., 2008 LC-UV-MS 42 pg mL -1 _(UV) 15 pg mL-1 (MS) Mafra Jr et al., 2009

RRLC-MS/MS C18 Empore disks 20-60 pg mL-1 De la Iglesia et al.,

2008

LC-MSn C18 SPE 30 pg mL-1 Wang et al., 2007

LC-MSn C18 SPE 5 pg mL Wang et al., 2012

TLC SAX-SPE 10 μg g-1 Zhao et al., 1997

Amino acid analysis 1 μg mL-1 Quilliam et al., 1998

Capillary Electrophoresis SCX / SAX SPE 0.15 μg g-1 Zhao et al., 1997

Table 2.1: Summary of literature values for the limits of detection a

HPLC+UV DAD refers to liquid chromatography with ultra violet diode array detection; HPLC+FD (FMOC) refers to liquid chromatography with fluorescence detection; AQC, NBD-F, and NBC-CL refers to the derivatisation agents used in each method; LC-MSn refers to liquid chromatography with multiple tandem mass spectrometry; LC-UV-MS refers to liquid chromatography ultra violet detection coupled with mass spectrometry; RRLC-MS/MS refers to rapid resolution liquid chromatography coupled with tandem mass spectrometry; TLC refers to thin layer chromatography. bSAX-SPE refers to strong anion exchange solid phase extraction; SCX refers to strong cation exchange solid phase extraction; C18 refers to octadecylsilane reversed phase sorbent material.

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