# Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review
## 4 Sample Preparation and Analytical Methods for Cyanotoxins and Prymnesins Detection
### Analytical Methods
#### 4.2.2 Mass Spectrometry  
Following sample extraction and clean-up, separation of compounds typically employs either liquid or gas chromatographic (LC or GC) techniques. Typically, LC methods use a reversed phase C18 or a hydrophilic interaction liquid chromatography (HILIC) column and either methanol:water or water:acetonitrile gradients for separation, as these allow for flexibility, speed, and adaptability to a wide range of detectors relying on UV absorbance, fluorescence, or mass spectrometry. GC can be used as a separation method for cyanotoxins [79]. However, many cyanotoxins, including MCs, are larger molecules and are either not volatile or do not ionize well without chemical derivatization techniques. In addition, GC-based methods might require a solvent-exchange step to a nonpolar solvent such as hexane or ethyl acetate prior to analysis, which also typically involves blowing samples down to dryness prior to reconstitution with the nonpolar solvent. As such, GC-based separation requires more complex and time-consuming sample preparation, while also presenting more avenues for toxin loss from samples. While there are examples in the literature of analytical methods for cyanotoxins using GC-MS methods, these are a clear minority [78]. The single example in Table 3 using GC-MS involved MMPB oxidation of MCs, and in this instance, using GC-MS as the detector required an added derivatization step in the workflow. Mass spectrometric methods for quantifying cyanotoxins rely on comparison with specific analytical standards for target compounds and can provide greater specificity than is possible with ELISA. In the LC-MS/MS techniques typically employed for toxin measurement, both a starting mass and a characteristic fragment ion specific to a given chemical are used to ensure specificity of assignment, along in some cases with a separate confirming ion fragment and a unique ratio of confirming to main ion. This is advantageous in that it means that false positive identifications are less common (although not impossible; ANA-a is isobaric with the common amino acid phenylalanine and both parent and product masses are identical; confusion of these two species can occur when MS-based detection is used [53]). An additional useful feature of LC-MS/MS methods is that surrogate and internal standards, usually consisting of isotopically labeled analogs of target compounds, can be added to samples prior to extraction and analysis. This enables the losses encountered during extraction, clean-up, and analysis steps to be accounted for through comparison with expected recoveries of surrogates, while isotopic dilution techniques can allow for compensation for some matrix interferences during all of these processes, improving overall quantitation accuracy [77]. Quantitative mass spectrometric methods have been developed for the majority of the cyanobacterial toxins, including MCs, ANA-a, CYNs, STXs, and BMAA, either as a native compound or after chemical modification [80]. This allows samples to be separated using conventional C18 phases, although with ANA-a(S), HILIC methods work well for the assessment of this very hydrophilic toxin [81]. Due to the specificity, sensitivity, and rapidity of the analysis, mass spectrometric methods are now the physicochemical method of choice for the quantitative analysis of most cyanobacterial toxins in complex matrices for labs with access to appropriate instrumentation. Cyanotoxins can also be detected by MS without preliminary chromatographic separation, particularly with time of flight (TOF) mass spectrometers in which many compounds can be identified and quantified concurrently. For example, matrix-assisted laser desorption/ionization coupled with TOF (MALDI-TOF) can be used to perform toxin analysis in even individual cell colonies [79]. In a typical workflow, target compounds enclosed in the dried and solid sample are ionized by a laser beam and accurately identified through the high mass resolution provided by the TOF instrument.