Oasis hlb

First, 4 mL of EDTA-McIlvaine buffer solution and 1 mL of acetonitrile were added to each sample in a 50 mL polypropylene tube and then agitated, sonicated, and centrifuged. The resulting supernatant was sieved through glass wool while being transferred to another 50 mL polypropylene tube. Then, 13 mL of EDTA-McIlvaine buffer solution were added to this tube, mixed, and passed through OASIS™ HLB^® (Waters Corp.) solid phase extraction columns. These columns were previously conditioned with 5 mL of methanol and 5 mL of HPLC-grade water. Afterwards, these columns were eluted using 5 mL of methanol, and the elute was evaporated under mild nitrogen flow in a water bath (45 ± 5 °C). Finally, samples were reconstituted in 200 μL of methanol and 300 μL of HPLC-grade water. This method was adapted from the methodology published by Berendsen et al. [54 (link)].

Methanol, acetonitrile, formic acid, ammonium formate, and water (all LC/MS or Optima grade) were obtained from Fisher Chemicals (Fair Lawn, NJ, USA). Ammonium hydroxide (NH₃ content 28–30%; analytical grade) was from Sigma-Aldrich (St. Louis, MO). Solid phase extraction (SPE) cartridges were 60-mg Oasis HLB with 30-µm particle size (Waters, Milford, MA). Borosilicate glass tubes, autosample vials, and polypropylene centrifuge tubes were purchased from Fisher Scientific. Milli Q water (>18 MΩ) was produced from a water purification system (Barnstead Nanopure). Multi-anion standards were from Sigma-Aldrich (St. Louis, MO, USA) and multi-cation standards were from Alltech (USA). Anatoxin-a, microcystin-LA, -LR, and -RR standards, as well as saxitoxin and cylindrospermopsin ELISA kits were all from Abraxis (Warminster, PA, USA). The 25-mm glass microfiber GF/A syringe filters were obtained from Whatman (GE Healthcare UK Limited, UK) and B-D 10-mL disposable syringes were from Becton Dickinson & Co (Franklin Lakes, NJ, USA). The IC eluents were sodium hydroxide (50%, w/w) for anion analysis and methanesulfonic acid for cation analysis.

The medulla oblongata and lumbar 4~5 spinal cord were removed from each animal and immediately stored in -80 ºC until use. Each tissue was lysed in lysis buffer (8 M urea in 1× PBS, pH 8.0, 1× Protease and Phosphatase Inhibitor [Thermo Scientific, New Jersey, USA]) and sonicated. The lysed sample was centrifuged at 12,000 g, 4 ºC for 30 min followed by collected the supernatant. BCA protein assay kit (Thermo Fisher, NJ, USA) was used to measure the concentration of extracted protein. For spinal cord samples, a total 100 μg of protein sample was pooled from equal portions of six individual mouse samples. The pooled sample was incubated with 10 mM dithiothreitol (DTT) for 1 h at 55 ºC followed by incubation with 25 mM iodoacetamide (IAA) for 1 h at room temperature (dark environment). Samples were then diluted to 1.0 M urea concentration with 1× PBS (pH 8.0) and digested with trypsin (1:25 w/w) (Promega, WI, USA) for 15 h at 37 °C. After digestion, the sample solution was adjusted to pH 1~2, the peptide mixture desalted with a reversed-phase column (Oasis HLB, Waters, USA) and the sample dried in a vacuum centrifuge for Tandem Mass Tag (TMT) labeling. For medullary tissue, 4 individual samples per group and 50 μg protein per sample were used for protein digestion and TMT labeling as for spinal cord samples.

Microcystin-LR (MC-LR) and nodularin (NOD) standards were purchased from Enzo Life Sciences (UK) Ltd and 2-Methoxy-4-phenylbutyric acid (MMPB) was purchased from Wako Pure Chemical Industries Ltd (Japan). All other chemicals used were purchased from Sigma Aldrich, UK, bar the OASIS HLB and OASIS PRiME HLB solid phase extraction (SPE) cartridges (60 mg, 3 cc) which were purchased from Waters, Ireland. The water used was supplied from an in-house 18 MΏ Millipore water system, Millipore (UK) Ltd.
Standards of MC-LR, NOD (IS) and MMPB used for method development, validation and sample analysis were prepared as outlined in the Supplementary Information online.

For the solid-phase extraction (SPE) procedure, the performance of three sorbents (3 mL, 500 mg) was compared. In detail, a strong cation exchanger resin, Bakerbond SCX aromatic sulfonic acid (Agilent), a mixed mode hydrophilic/lipophilic polymer based resin, OASIS HLB (Waters) and a silica gel sorbent, Discovery SPE Pure Silica (Supelco), were used.
Each cartridge was conditioned with different solutions, depending on the type of adsorbent: (i) SCX was conditioned with 3 mL of water, followed by 3 mL of water acidified to pH 2 with HCl; (ii) HLB with 3 mL of methanol followed by 3 mL of water, while (iii) pure silica with 5 mL of methanol.
For each cartridge, aliquots of 4-mL solutions containing 50 μg/L of betaine were loaded with a flow rate of 1–2 mL/min flow rate. The eluents tested in the recovery step were chosen according to the expected analyte–sorbent interactions. Before elution, each cartridge was washed with 2 mL of the proper solvent (H₂O acidified to pH 0.5 for SCX cartridge, H₂O for polymeric sorbent and methanol for pure silica resin) to remove unretained compounds. To check the retention of the analyte, the following fractions were collected during each step of the SPE protocol and then analysed by HPLC-MS/MS: (i) the solution after loading, (ii) the washing solution and (iii) the eluate.