1100 liquid chromatograph

Overnight dark-adapted wild-type (n=4) and AdipoR1^−/− (n=4) mice (3–4 months of age) were anaesthetized with an intraperitoneal injection of ketamine and xylazine. Pupils were dilated with 1% (w/v) atropine sulfate in saline solution. Two mice of each genotype were exposed to light at 1,000 lux for 10 min; the remaining mice were maintained in darkness. Mice were killed by cervical dislocation under anaesthesia and eyes enucleated. After removing the anterior segment, eyecups were homogenized in 20 mM HEPES buffer containing 0.1% SDS and hydroxylamine, and retinoids extracted with hexane under dim red light (Kodak Wratten 1A). Total retinoids, all-trans-retinyl esters and 11-cis retinaldehyde from the dark-adapted and light-exposed mice were measured by normal-phase HPLC (Agilent 1100 liquid chromatograph) equipped with a ultraviolet photodiode-array detector. Retinoids in the samples were separated by gradient elution of the mobile phase (0.2–10% dioxane in hexane, 2 ml min⁻¹ flow rate) on a silica column (Zorbax-Sil 5 μm, 250 × 4.6 mm, Agilent Technologies). Identified peaks were confirmed by spectral analysis and co-elution with authentic retinoid standards.

Citrinin was determined using high-performance liquid chromatography, HPLC (Agilent 1100 liquid chromatograph) with a diode array detector (DAD) and an autosampler. The separation was completed using a Discovery C18 column (5 μm, 150 × 4.6 mm column) (Supelco Inc., Bellefonte, PA, USA) and an isocratic elution. The mobile phase consisted of acetonitrile:water containing 0.05% TFA and the volume ratio was 35:65 [50 (link),51 ]. All samples, standards, and solvents were filtered through 0.22 μm membrane filters prior to HPLC analysis. The flow rate was 1 mL/min, and 20 μL sample was injected. The UV-DAD detection was monitored at 254 nm and 334 nm. The integrated peak areas at 334 nm for standard citrinin were used to determine a 7-point calibration curve and used to extrapolate citrinin recovered. The LOD and LOQ for the experimental conditions were 1.11 μg/mL and 3.70 μg/mL, respectively. Recovery of citrinin was determined by dividing citrinin concentration recovered by known citrinin concentration injected.

Samples were analyzed using an Agilent Technologies 1100 liquid chromatograph (Santa Clara, CA, USA) equipped with an automatic injector, degasser system, quaternary pump with four solvent channels, a column thermostat, and fluorescence. The chromatographic separation of sulfonamides was performed using a method previously described by Patyra et al. [19 (link)]. The Zorbax Eclipse XDB (150 × 4.6 mm, 5 µm) column from Agilent Technologies (Santa Clara, CA, USA) protected by a RP18 guard column (4.0 × 3.0 mm, 5 µm) from Phenomenex (Torrance, CA, USA) was used for sulfonamides separation. The mobile phase consisted of 0.08% acetic acid in water (v/v; phase A), acetonitrile (phase B), and methanol (phase C). The gradient profile is shown in Table 4. The flow rate was 0.6 mL/min and the column thermostat was set at 25 °C. The injection volume was 40 μL. The excitation and emission wavelengths for all analyzed sulfonamides were 405 and 495 nm, respectively, and total run time was 27 minutes for each injection.