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Conventional multivariable optimization is usually based on the ��one-factor-at-a-time�� approach, which is unable to detect interactions among independent variables and presents a lack of complete information on the effects of all determinants [21]. Response surface methodology (RSM) is a useful tool for evaluating multiple parameters and their interactions based on quantitative data that may effectively overcome the drawbacks of classic ��one-factor-at-a-time�� or ��full-factors�� approaches [18]. The aim of this study is to develop a simple but effective extraction method for simultaneous determination of four TCs residues in animal manure through HPLC. Here, UAE conditions were investigated. Factors including ultrasonic temperature, solution volume, and pH were optimized by using RSM. The optimal Isotretinoin experimental parameters were validated by real samples. 2. Materials and Methods 2.1. Reagents and Materials HPLC-grade methanol (MeOH) and acetonitrile (MeCN) were obtained from Merck (Darmstadt, Germany). Distilled water was purified to ultrapure water in a Milli-Q system (Millipore, USA). OTC, TC, CTC, and DC were purchased from Dr. Ehrenstorfer (Augsburg, Germany). All other chemical reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Cell Cycle inhibitor Co., Ltd. (Shanghai, China). The McIlvaine buffer used in this work was composed of 0.2?M citric acid and 0.4?M?Na2HPO4 (3?:?2,?v/v). A mixed solution consisting of McIlvaine buffer, 0.1?M?Na2EDTA solution, and MeOH at a ratio of 25?:?25?:?50 (v/v/v) was selected as the extraction buffer, similar to previous studies [23�C27]. pH was adjusted by addition of H3PO4 or NaOH. 2.2. Standard Solutions and Samples Individual stock solutions (2.0?mg/mL) were prepared by dissolving each TCs standard in MeOH. Working mixed standard solutions were prepared by mixing appropriate amounts of the four TCs solutions above to obtain a concentration of 500?��g/mL and diluting with MeOH to 100?��g/mL. These solutions were Kinase Inhibitor Library datasheet stored in amber glass bottles at 4��C. Cow manure samples were supplied by Agro-tech Extension Center in Pudong District, Shanghai. After air-drying and grinding, samples were sieved to