Chromnav2

The integrated HPLC system (Jasco, Tokyo, Japan) used contained an autosampler (AS-4050), a binary pump (PU-4180), and a fluorescence detector (FP-920). Chromatographic data were evaluated employing ChromNAV2 software (Jasco, Tokyo, Japan). The effects of Mg²⁺ on the sensitivity regarding the HPLC-FLD analyses of AOH were tested employing the following two methods. In the first HPLC method, a SecurityGuard guard column (C8, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, USA) was linked to a Kinetex C8 (100 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) analytical column; while in the second method, a SecurityGuard guard column (C18, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, US) was linked to a Gemini C18 (150 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, US) chromatographic column. In both assays, the mobile phase consisted of HEPES buffer (10 mM, pH 7.0) and acetonitrile (70:30 v/v%), without or with MgCl₂ (50 mM). The injected sample volumes were 20 μL, the analyses were carried out at 1.0 mL/min flow rate and room temperature. AOH was detected at 455 nm emission wavelength (λ_ex = 345 nm). The linearity was tested between 0 and 1000 nM mycotoxin concentrations. LOD and LOQ values were determined as the lowest concentrations where signal-to-noise ratios were 3 and 10, respectively. Intra-day repeatability was tested only for the Mg²⁺-sensitized methods (n = 7).

Measurement of gemcitabine in different samples was done by high performance liquid chromatography (HPLC) system (LC4000+ Series, Jasco, Tokyo, Japan). A HPLC system consists of LiChrospher 100 C18 column (4.6 × 250 mm, particle size 5 µm) with a 20 µL sample loop connected to UV-Visible detector (MD-4010, Jasco, Tokyo, Japan) and a software for data acquisition (ChromNAV 2.0, Jasco, Tokyo, Japan) was used. Elution of gemcitabine was achieved by a mobile phase consisting of acetonitrile and acetate buffer (97:3% v/v; pH 5). The column temperature was maintained at 25 °C, while the flow rate of mobile phase was 1 mL/min. Samples (20 µL) were injected and the detection was done at 280 nm [44 (link)]. Linear regression analysis indicates good linearity in the concentration of 25–800 ng/mL (r² = 0.995) and the retention time was estimated as 3.54 min. The LOQ (limit of quantitation) and LOD (limit of detection) of this method are 18.90 ng/mL and 6.23 ng/mL, respectively. The coefficient of variation and the accuracy range were 1.89%–8.25% and −2.67–9.05, respectively.

GLU and GABA levels in the NAc and VTA were assessed under the different experimental conditions. An aliquot (20 μL) of the filtrate was injected into the HPLC–fluorometer and the determination of GLU and GABA was performed as described previously [62 (link)]. Briefly, 20 μL of the sample were mixed with 4 μL of borate buffer (pH 10.8), and then the mixture was derivatized by adding 4 μL of fluorogenic reagent (20 mg of orthophthaldehyde and 10 μL of β-mercaptoethanol in 5 mL of ethanol). Next, 90 s after derivatization, samples were injected into a HPLC system with the following configuration: isocratic pump (model PU-4180, Jasco Co., Ltd., Tokyo, Japan), a C-18 reverse phase column (Kromasil 3-4.6, Bohus, Sweden), and a fluorescence detector (model FP-4020, Jasco Co., Ltd., Tokyo, Japan). The mobile phase containing 0.1 M NaH₂PO₄ and 24.0% (v/v) CH₃CN (pH adjusted to 5.7) was pumped at a flow rate of 0.8 mL/min. The retention time for glutamate was 1.8 min and for GABA was 11 min while the detection limit was 5 fmol/μL. Dialysate samples were analyzed by comparing the peak area and elution time with reference standards (ChromNAV 2.0, Jasco Co., Ltd., Tokyo, Japan).

Quantification of moxifloxacin was performed using high-performance liquid chromatography (HPLC) system (PU 2080, UV– 2075 plus, Jasco, Tokyo, Japan). The HPLC system utilized is made of Phenomenex C-18 column (150 × 4.6 mm, i.d 5 μm) connected to UV-Visible detector (MD-4010) and a software for data acquisition (ChromNAV 2.0, Jasco, Tokyo, Japan). Chromatographic separation of moxifloxacin was accomplished using a mixture of mobile phase consist of acetonitrile: potassium dihydrogen ortho-phosphate (0.02 mM) 20:80 v/v, adjusted to a pH 4.5 with phosphoric acid [19 (link)]. The temperature in the C-18 column was set at 25°C, while the rate of solvent flow was fixed at 1 ml/min to elute moxifloxacin and was detected at 305 nm. Regression analysis indicates good linearity when moxifloxacin concentration was in the range of 25–300 ng/ml (r² = 0.995).