# Presence of Cyanotoxins in a Mexican Subtropical Monometric Crater Lake
## 2 Materials and Methods
### Extraction and Analysis  
#### 2.4.1 Microcystin Extraction  
Each filter was placed into a separate 50 mL polypropylene centrifuge tube with 20 mL of MeOH and agitated for one hour. The MeOH fraction was recovered and transferred into a new 50 mL tube. This procedure was repeated three times. The recovered extract was filtered through a syringe filter of 25 mm with a 0.45 \(\upmu\)m pore size. MeOH was rotary evaporated and, the dry residues were resuspended  
Figure 2: Location of Santa Maria del Oro lake in the state of Nayarit (black point in the left figure) and sampling stations (numbers in the right figure). in 1 mL of MeOH (grade HPLC) and placed in a sealed glass HPLC vial. For net samples, a known amount of the dry mat (between 0.5 g and 1 g) was treated following the same procedure for the filters, with the exception that the re-extraction that was performed six times because the samples were much more concentrated. UHPLC-MS/MS and Microcystin Quantitation  
The UHPLC-MS/MS analysis was conducted following the method of [PERSON] et al. (2013), as applied previously to the routine analysis of water and algae in England (2016). A Waters (Manchester, UK) Xevo TQ tandem quadrupole mass spectrometer (MS/MS), coupled with a Waters Acquity UHPLC, was used for LC-MS/MS analysis. Chromatography was conducted using a 1.7 \(\upmu\)m, 2.1 \(\times\) 50 mm Waters Acquity UHPLC BEH C18 column (P/N 186002350, Lot no. 0249343351) in conjunction with a Waters VanGuard BEH C18 1.7 \(\upmu\)m, 2.1 \(\times\) 5 mm guard cartridge (P/N 186003975, Lot no. 0245343321). The columns were held at +60 \({}^{\circ}\)C, with samples held in the sample manager at +4 \({}^{\circ}\)C. Mobile phase A1 consisted of water + 0.025% formic acid, and mobile phase B1 comprised of acetonitrile (MeCN) + 0.025% formic acid. The sample injection volume was 5 \(\upmu\)L, and the mobile phase flow rate was consistently 0.6 mL/min. The UPLC gradient was exactly as described by [PERSON] et al. (2013), with a total run time of 5.5 min. Each instrumental sequence started with a series of instrumental blanks, followed by toxin calibration standards and an extract of RM-BGA to be used as a matrix-based retention time marker, and as internal quality control (QC). The Waters Xevo TQ tune parameters were as follows: 150 \({}^{\circ}\)C source temperature, 600 \({}^{\circ}\)C desolvation temperature, 600 L/h desolvation gas flow and 0.15 mL/min collision gas flow. Capillary voltage was held at 1.0 kV. Selected reaction monitoring (SRM) transitions were built into the MS/MS method, using positive mode acquisition for each toxin. Parent and daughter ions, all in positive ion mode, as well as cone and collision voltages are summarized (Table 1). The majority of toxins exhibited unique SRM transitions and chromatographic retention times, resulting in good separation of cyanotoxins over the 5 min run time. The exceptions were [Dha7]-MC-LR and Asp3-MC-LR, which shared the same transitions and could not be completely resolved. These two analytes are therefore reported together. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Analyte** & **SRM Transitions** & **Cone, V** & **CE, eV** \\ \hline MC-RR & 519.9 \(>\) 134.9; 126.9; 102.8 & 30 & 30; 50; 70 \\ Nod & 825.5 \(>\) 135.1; 103.1 & 55 & 60; 100 \\ MC-LA & 910.1 \(>\) 135.1; 106.9 & 35 & 70; 80 \\ [Dha7]-MC-LR & 981.5 \(>\) 135.0; 106.8 & 75 & 75; 80 \\ Asp3-MC-LR & 981.5 \(>\) 134.9; 106.9 & 75 & 70; 80 \\ MC-LF & 986.5 \(>\) 213.0; 135.0 & 35 & 60; 65 \\ MC-LR & 995.6 \(>\) 135.0; 127.0 & 60 & 70; 90 \\ MC-LY & 1002.5 \(>\) 135.0; 106.9 & 40 & 70; 90 \\ MC-HiilR & 1009.7 \(>\) 134.9; 126.9; 106.9 & 75 & 75; 90; 80 \\ MC-LW & 1025.5 \(>\) 134.9; 126.8 & 35 & 65; 90 \\ MC-YR & 1045.6 \(>\) 135.0; 126.9 & 75 & 75; 90 \\ MC-HtyR & 1059.6 \(>\) 134.9; 106.9 & 75 & 70; 90 \\ MC-WR & 1068.6 \(>\) 134.9; 106.9 & 80 & 75; 100 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Selected reaction monitoring (SRM) transitions used for microcystin (MC) detection and quantitation.