Ultrapure water was produced using a Barnstead Nanopure water system for all aqueous solutions. All chemicals and solvents were of ACS reagent grade. Standard stock solutions of 1000 mg/L alachlor and 2,6 DEA were prepared by dissolving 0. 100 g of alachlor and 2,6 DEA individually in 90 mL of HPLC grade methanol and diluted to 100 mL, the solutions were stored at 4 C in silanized brown glass bottles with Teflon lined caps. Fresh working standard solutions were prepared daily by appropriately diluting the stock solution to the studied concentrations with water. The chemical structures of alachlor and its metabolite 2,6 DEA are shown in Figure 1a. Sodium dihydrogen phosphate and sodium hydroxide were obtained from Riedel deHa?en to prepare buffer solution for adjust the pH.

The mobile LY-411575 phase was prepared as 50% of acetonitrile in water containing 0. 01 M phosphate buffer at pH 7. 0. All eluents were filtered through a 0. 45 m poly membrane filter and degassed ultrasonically. Apparatus and Instrumentation. HPLC analyses were per formed using a Dynamax liquid chromatograph system equipped with a Dynamax SD 200 solvent delivery system and a Dynamax UV 1 detector with a 20 L flow cell. Separations were performed on an LC 18 column, 5 m particle size). The Varian Star chromatography workstation was utilized to perform HPLC operations to obtain the chromatogram and to carry out data calculations. A Rheodyne 7010 injector/switching valve with a 20 L sample loop was used as the interface between the HF LPME system and the HPLC system for sample introduction.

The hollow fiber microdialysis system comprised a microinjection syringe pump and its controller and a 1 mL syringe. A cellulose acetate hollow fiber membrane was purchased from Baxter/Althin Co.. A regenerated cellulose acetate hollow fiber membrane was obtained from Spectrum Laboratories Inc.. A home assembled hollow MEK Signaling Pathway fiber membrane probe was prepared and utilized as the HF LPME enriched sampling system. By using polyethylene tubings, the inlet of the hollow fiber for LPME was connected to a syringe pump containing perfusion solvent and the outlet connected to the sample loop of a switching valve. The online micro dialysis sampling coupled HPLC was assembled with minor modifica tions as in our previous paper. 29,32,34 A schematic diagram of the online HF LPME/HPLC UV system used for determination of alachlor and 2,6 DEA in culture medium is presented in Figure 1b.

Preparation of Culture Media and Spiked Samples. Nu trient agar culture medium was prepared by adding 2. 3 g of NA and 1. 5 g of agar in a 200 mL flask containing 90 mL of water. After dilution to 100 GPCR Signaling mL with water, the NA culture medium was sterilized in an autoclave for 1 h. After cooling in a clean bench, the culture medium was transferred into 125 mL culture medium flasks. Alachlor and 2,6 DEA were spiked in the culture medium as spiked samples. Potato dextrose broth culture medium was prepared by adding 24 g of PDB in a 1000 mL ask containing 900 mL of water. After dilution to 1000 mL with water, the culture medium was transferred into 125 mL asks and sterilized in an autoclave for 1 h. Rhizopus stolonifer with a concentration of 5.

0 _ 5 10 spores/mL was incubated at 28 C in PDB culture medium DNA Damage for 96 h to degrade alachlor. Online HF LPME/HPLC UV Procedure. After appropriate dilu tion or adjustment, culture medium containing alachlor and 2,6 DEA was transferred into the dialysis cell for HF LPME by using hexane as perfusion solvent at 0. 1_4 L/min flow rate. The dialysate was collected online in the sample loop for HPLC analysis. The experiments for each investigation were carried out with five replicates. The enrichment factor in online HF LPME is calculated on the basis of the ratio of analyte concentration in the extractant. Optimization of Chromatographic Conditions. Because the online HF LPME system was a pretreatment step for the HPLC determination, in addition to optimizing the pretreatment sys tem, chromatographic conditions had to be optimized and built up.

When referred to the literature, a reversed phase C 18 column had the potential to resolve alachlor and 2,6 DEA from other species and was thus tested herein. From a series of tests, the optimum separation and detection conditions were achieved. Under the optimum conditions acetonitrile in 0. 01 M phosphate aqueous buffer NSCLC at pH 7. 0 at the flow rate of 1 mL/min), peaks of alachlor and 2,6 DEA in the chromatogram were sharp and symmetric and well separated within 18 min. On the basis of the UV spectra of alachlor and 2,6 DEA in elution solution, the wavelength for UV detection was set at 210 nm. Under the chromatographic conditions, the retention times of alachlor and 2,6 DEA were 17. 18 min and 12. 60 min, respectively, and the reproducibilities of quantitative detection were 2. 78 and 1. 23% RSD for three deter minations of alachlor and 2,6 DEA, respectively. Selection of Hollow Fiber.