Tcc 3000sd

The residues of dimethoate and chlorpyrifos were quantified using High-Performance Liquid Chromatography (HPLC) coupled with a photo-diode array (PDA) detector. The HPLC was made by Dionex and has the following components: column compartment TCC-3000 SD, photo-diode array detector DAD-3000, autosampler ACC- 3000 T, and quaternary pumps LPG-3400 SD. The C18 column (Phenomenexkinetex 250 4.6 mm, 5 m, 100 A) was used for the separation. Acetonitrile and water (9:1 v/v) were used as the mobile phase, with UV detection taking place at 205 nm and a flow rate of 1 ml min⁻¹. Column compartment temperature was kept at 25 °C. The total run time was 10 min. Under these operating conditions, dimethoate and chlorpyrifos retention times were 3.05 and 4.23 min, respectively. Data analysis was performed by chromeleon software 6.80 version.
The following formula quantified the quantification of insecticides recovered from the muscles of the fish:
$\mathrm{Insecticide}(\mathrm{ppm})\frac{\mathrm{Standard\; injected}(\mathrm{ng})}{\mathit{Standar\; area}}x\frac{\mathit{Sample\; area}}{\mathit{Sample\; injected}(\mathrm{\mu l})}x\frac{\mathit{Sample\; volume}}{\mathit{Sample\; weight}(\mathrm{g})}$
Bio-concentration factor (BCF) was calculated by the following formula [15] .
$\mathit{BCF}(\mathit{L\; per}\mathrm{kg})\frac{\mathrm{The\; concentration\; of\; the\; substance\; in\; fish}(\mathrm{mg\; per\; kg})}{\mathrm{The\; concentration\; of\; the\; substance\; in\; water}(\mathrm{mg\; per\; L})}$

HPLC analysis was carried out on a Thermo Scientific Dionex UltiMate 3000 (Dionex Corporation, Sunnyvale, CA, USA) equipped with a pump LPG-3400SD, column oven TCC-3000SD, detector DAD-3000 and autosampler WPS-3000TSL. Chromatographic separations were carried out using column RP-18 LiChroCART, 125 mm × 3 mm, 5 µm (Merck, Darmstadt, Germany) at a temperature of 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was 1.0 mL/min with the following gradient elution: starting at 7% mobile phase B and holding for 0.5 min, reaching 50% in 4 min and 95% in 4.5 min and holding for 1 min. From 5.5 min, the gradient returned to 7% mobile phase B and stopped after 7 min in this concentration. The injection volume of the sample was 10 µL, and detection was performed at 280 nm wavelength. The commercial TC of various concentrations was used to prepare the calibration curve (y = 0.2625x − 0.0771, R² = 0.998).

Withanolide extraction and the analytical HPLC experiments were determined according to a protocol previously published by Takshak and Agrawal (2014). Five additional calibration levels of each standard compound (withanolide A, withanolide, and withaferin A) were prepared by diluting the stock solutions with concentrations ranging from 0 to 100 ppm. Separation of withanolides was performed by HPLC; 10 µL of each sample was injected using a WPS-3000-SL Dionex Semi Prep Autosampler (Dionex Corporation, Sunnyvale, CA, USA). All sampling was carried out with a UV-DAD detector (MWD-3100 Dionex UV-VIS Detector) operating at 229 nm, with a flow rate of 0.6 mL/min (binary pump, LPG 3400SD Dionex) at 30 °C (column oven system, TCC3000SD, Dionex) and an Inertsil ODS-3 (GL Sciences Inc., Tokyo, Japan) column (150 × 4.6 mm). The isocratic flow was performed using methanol/water containing 1% trifluoroacetic acid (w/v) in a gradient fashion (45:55–65:35) for 15 min (Grover et al., 2013).

Organic acids were detected in the tested plants by using HPLC under the following conditions: 0.001 N sulfuric acid, at 210 nm, 0.6 mL min¹. The HPLC system consisted of a liquid chromatographer (Dionex, Sunnyvale, CA, USA) and a detector (LED, ultimate 3000), in addition to a pump (LPG-3400A), a column thermostat (TCC-3000SD), and an autosampler (EWPS-3000SI). Separation of organic acids was conducted through an Aminex HPH-87 H (300 × 7.8 mm) column coupled with IG Cation H (30 × 4.6) precolumn of the Com-Red firm (at 65 °C). Data analysis and interpretation were performed using chromeleon v.6.8 computer software [21 (link)].

The sample analysis was performed by using a Dionex UltiMate 3000 UHPLC system (Thermo Scientific, San Jose, CA, USA), comprising a LPG-3400SD binary pump, WPS-3000TSL thermostat autosampler, TCC-3000SD thermostat column compartment and DAD-3000 diode array detector. The data were recorded and analyzed by Chromeleon™ 7.2 Chromatography Data System Software (Thermo Scientific, San Jose, CA, USA).
The chromatographic separation of MI, MCI, MP, EP, PP and BP was achieved through an ACCLAIM™ 120 C₈ analytical column with the dimensions 150 mm × 2.1 mm and 5 μm of particles size (Thermo Scientific, San Jose, USA). The optimal separation was obtained by using binary mobile phase: water (0.1% trifluoroacetic acid, pH 2.1, solvent A) and acetonitrile (solvent B) at a flow rate of 0.5 mL/min. The gradient mobile phase elution was 0–2 min (B, 12.5%), 2–4 min (B 20–30%), 4–16 min (B, 30–50%), 16–22 min (B, 50–100%), return to its equilibrium conditions and 22–30 min. The column temperature was kept at 35 °C, and the sample injection volume was 10 µL. The column was also washed with a mixture (50:50, v/v) of methanol and Milli-Q water solution, for five minutes, following the analysis of every ten samples. The optimal detection wavelength was performed in the UV range at 255 nm.