2475 flr detector

HPLC analysis was to detect the contents of DA and dihydroxy-phenyl acetic acid (DOPAC) in the striatum of mice. Briefly, the striatum were rapidly dissected and sonicated. After mixture with borate buffer, AccQ-Fluor reagent (WAT052880, Waters, Milford, USA) was added to 100 μl. Samples were then centrifuged for 30 min at 12,000 g, and the supernatants were finally filtered by 0.22 μm microfilters (Millex, Barcelona, Spain) before analyzing by HPLC. In this study, The Waters HPLC included a system controller (600E, Waters), a quaternary pump (2535, Waters), a fluorescence detector (2475 FLR Detector, Waters), and an autosampler (2707 Autosampler, Waters). Millenium software (Waters) was applied for collecting data, calculating peak area values, and controlling the system. The silica particle C8 Symmetry column (5 μm) with a 100-Å pore (Waters) was used for separation. The mobile phase included EDTA-2H₂O (pH 2.65, 0.027 mM), NaH₂PO₄ (0.1 M), potassium chloride (2 mM), 18% methanol, and octyl sodium sulphate (1 mM). The flow rate of mobile phase was set at 1.0 ml/min. Fluorescence excitation was set at 252 nm, while the emission was determined at 397 nm.

UPLC-FLD analysis was performed on a Waters e2695 HPLC system (Palo Alto, Santa Clara, CA, USA) coupled to a programmable fluorescence detector (Waters 2475 FLR Detector). The sample extract was injected onto a Waters HSS T3 column (100 × 2.1 mm, 1.8 µm, 5 μL injection volume) at 40 °C for chromatographic separation. The column was eluted with a binary solvent system of water (A) and acetonitrile (B). Gradient elution at constant flow rate of 0.2 mL/min was used. The solvent gradient started from 30% B and was programmed to linearly increase to 100% B in 12 min, hold for another 5 min, and then recondition at 30% B. The eluate was monitored by FLD, which was time-programmed at excitation/emission wavelengths as follows: 0–10 min: λ_ex 240 and λ_em 435 nm (for 1-aminopyrene); 10–20 min: λ_ex 240 and λ_em 390 nm (for pyrene). As shown in Figure 2, the 1-aminopyrene eluted at 9.42 min, and pyrene eluted at 11.35 min.

At 30 h, 100 µL of liquid medium from each dish (n = 9) per biological rep was added to HPLC grade methanol. At 72 and 96 h, 500 µL of medium from each biological replicate (a single Petri dish) was added to 500 µL of HPLC grade methanol. Extracts were filtered through 200 mg basic alumina (58Å, 60-mesh powder, 11503-A1, Alfa Aesar, Tewksbury MA) in 1.5 mL polypropylene columns with 20 µm polyethylene frits [68 (link)]. 10 µL of each sample was separated in a Waters e2695 Separations Model HPLC (Waters Corp., Milford, MA) using a Nova-Pak C18 4 µm, 3.9 mm × 150 mm column held at 38 °C with an isocratic solvent system (37.5 Methanol: 62.5 water at a 0.8 mL/min) coupled to a PHRED photochemical reactor cell (Aura Industries Inc., New York, NY, USA). After separation and photolytic derivatization, a 2475 FLR Detector (Waters Corp., Milford, MA, USA) was used to detect and quantify aflatoxin B₁ (365 nm Ex, 440 nm Em) [69 (link),70 (link)]. Run time was 17 min with aflatoxin B₁ peak eluting at~13.5 min. Empower software (Waters Corp., Milford, MA, USA) was used to integrate the aflatoxin B₁ peak. Aflatoxin quantity was calculated based on a calibration curve calculated from 4 replicates of standards with 1, 5, 50, 500 and 1000 ng/mL aflatoxin B₁ [70 (link)]. Aflatoxin B₁ minimum level of detection was <0.05 ppb and minimum quantification from standard curve was 1 ppb.