Spd 10av detector

The quantitative determination of ETZ in the receiving fluid and in skin samples was determined by a reverse-phase HPLC method with apparatus consisting of an LC-10AD pump and 20 μL Rheodyne injector, SPD-10AV detector and C-R4A computer integrating system (Shimadzu Corp., Kyoto, Japan). A reverse-phase C18 column (Synergi 4u Fusion-RP 80A, 150 × 4.6 mm, Phenomenex, Torrance, CA, USA) was used. Isocratic elution was performed using a mobile phase consisting of a mixture of CH₃OH:H₂O acidified with 1% glacial acetic acid (98:2 v/v), filtered through a 0.45 μm pore size membrane filter. The detection wavelength was 310 nm, the flux was 1.0 mL/min and the retention time was 11.3 min.
The amount of ETZ in the samples was determined by comparison with appropriate external standard curves obtained applying the least square linear regression analysis. For in vitro studies, the calibration curves were obtained by dissolving the ETZ in acetonitrile and then diluting with PBS pH 7.4 added to 0.01% Brij 98. In the case of biological materials, a standard curve was obtained by adding increasing amounts of ETZ to a blank biological matrix.

Nucleotide content was estimated as described by Jung et al. [21 (link)], with slight changes. A minced meat sample (5 g) of each chicken was mixed with 20 mL of 5% (volume/volume) perchloric acid to extract nucleic acids. Extracted nucleic acids were centrifuged at 9200× g for 10 min. The supernatant was then adjusted to pH 6.4 with 1 mol/L KOH. The supernatant was placed in a volumetric flask and adjusted to a volume of 25 mL with distilled water, filtered through a 0.22 μm membrane filter, and analyzed for adenosine triphosphate (ATP), and its related compounds were measured by HPLC (Shimadzu, Kyotos, Japan) equipped with an SPD-10A (V) detector, VP-CDS C18 column (4.6 mm id × 250 mm, 5 μm). The sample (10 μL) was injected at a flow rate of 0.7 mL/min, and the peak was detected at 254 nm. The amounts of ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP), IMP, inosine (HxR), hypoxanthine (Hx), and GMP were determined and calculated based on the standard ATP, ADP, AMP, IMP, HxR, Hx, and GMP. All standards reagents were purchased from Sigma (Merck, Darmstadt, Germany). The results were expressed as milligram of nucleotides contents per 100 g of wet muscle tissue.

Total acidity (TA) was determined according to AOAC official titrimetric method (AOAC method 982.27) [23 ] and pH was measured with a MP220 pH Meter (Mettler-Toledo, Greifensee, Zurich, Switzerland). Chromatographic analysis of individual organic acids was carried out using an HPLC chromatographic system composed of an LC-20AD pump and a UV/Vis SPD-10AV detector (Shimadzu, Kyoto, Japan). Separation was achieved on a cation exchange resin-based column Agilent Hi-plex H with an aqueous 10 mM sulphuric acid solution and a flow rate of 0.6 mL/min. The oven temperature was 50 °C and the injected volume was 10 µL. The Clarity Chromatography Software v8.2 (DataApex, Prague, Czech Republic) was used for data processing. The monitoring wavelength was set at 210 nm. Peak identification was carried out by comparison of their retention times with those of standard compounds. External standard calibration curves were prepared using solutions of citric, tartaric, malic, and acetic acids in the range of 0.5–5.0 g/L for quantification of each compound, and the results were expressed as g/L of PJ.