Sil 10ad autosampler

Chromatographic separations of amino acids were performed on a Shimadzu HPLC system consisting of two LC-20AD isocratic pumps, a DGU-14A degasser, an SIL-10AD autosampler and a fluorescence detector (RF-535) (Shimadzu, Tokyo, Japan). The analytical column was a reversed phase LC-C₁₈ DB (250 × 4.6 mm, 5.0 μm, Supelco (Bellefonte, United States). LC-solution^® software version 1.25 SP4 was utilized for hardware control and data manipulation. All separations were accomplished using a binary gradient elution program. The mobile phases A and B were 20 mM phosphate buffer (pH 2.8) and methanol, respectively. Their initial ratio was 40% v/v of B and the flow rate was set at 0.7 mL min⁻¹. The ratio of the mobile phase B was linearly increased to 65% in 15 min and kept constant for 9 min. Then, it was further increased to 80% in 1 min, while the flow was decreased at 0.6 mL min⁻¹ and stayed constant for up to 30 min. Then, it reverted to its initial conditions (40% B and flow 0.7 mL min⁻¹) in 5 min and kept constant for up to 40 min to obtain reproducible separations. The injection volume was set at 20 μL (for the analysis of the product) and 100 μL (to determine the LOD value on the chocolate substrate). The column was thermostated at 25 °C. Amino acid-OPA derivatives were detected spectrofluorimetrically at_λex/λem = 340/455 nm.

HPLC analyses were performed using a Shimadzu HPLC system equipped with an SCL-10A controller, LC-10 AD binary solvent delivery pumps, SIL-10 AD autosampler, SPD-M10 A diode array detector, and an RF-10 A XL fluorescence detector. An Alltech 5 μm particle size column was used with a 4.6 x 150 mm reverse phase octadecylsilica (C18). To monitor the chromatogram, the RF fluorescence detector was set at 321 nm for excitation and 465 nm for emission and the SPD diode array detector was set at 400 nm. HPLC grade solvents were prepared as follows: solvent A, 0.1% TFA in water and solvent B, 0.1% TFA in 80% acetonitrile. Solvent gradients were set as follows: 0–10 min 55–65% B, 10–14 min 65–90% B, followed by reducing solvent B composition to 55% within 14–24 min. The column elution was carried out at a flow rate of 0.8 ml/min with a linear gradient of solvents. After treatment of MLT with MPO in presence of H₂O₂ for 24 hours, the reaction mixture was filtered through an Amicon Ultra-15 centrifugal filter unit with Ultracel-10 membrane (from Millipore) 3-kDa cut-off by centrifuging at 14,000 rcf rate for 30 min at 4°C; then 50 μl of filtered sample was injected for analysis. At the end of the run the system was equilibrated with 45% solvent A; each sample was analyzed in triplicate.

Chromatographic separation of homotaurine and histidine was performed using a Shimadzu HPLC system consisting of two LC-20AD isocratic pumps, a DGU-14A degasser, an SIL-10AD autosampler and a fluorescence detector (RF-535, Shimadzu, Tokyo, Japan). The stationary phase was a reversed-phase LC-C₁₈ DB column (250 × 4.6 mm, 5.0 μm), Supelco (Bellefonte, PA, USA), thermostated at 30 °C. LC-solution^® software version 1.25 SP4 was used for hardware control and data manipulation. All separations were performed using a binary gradient elution program. Mobile phases A and B consisted of potassium phosphate buffer (pH = 7; 0.02 M) and methanol, respectively. The injection volume was set at 40 µL and the flow rate was set at 1 mL·min⁻¹. Homotaurine and histidine OPA derivatives were monitored at λ_ext = 340 and λ_em = 455 nm, respectively.