Lc ms grade methanol

A Milli-Q water purification system (Millipore, MA, USA) was used in the preparation of deionized water (18.2 MΩ). Acetonitrile and chloroform were HPLC grade purchased from Fischer Scientific (Loughborough, UK). Methanol (LC–MS grade) and ammonium acetate were purchased from Sigma-Aldrich (Dorset, UK). Isopropanol (LC–MS grade) was obtained from Fischer Scientific (Loughborough, UK).

2,4,6-Trinitrobenzenesulfonic acid was obtained from Sigma-Aldrich (St. Louis, MO, USA). IL-17 and PGE₂ ELISA kits were purchased from Shanghai Panke Industrial Co., Ltd (Shanghai, China). The RNA PCR Kit was sourced from Takara (Dalian, China). Formic acid (LC-MS grade) was purchased from Tedia Company Inc. (Fairfield, OH, USA). Acetonitrile and methanol (LC-MS grade) were purchased from Sigma-Aldrich.

The chemicals, including the standard IS potassium salt (purity 99.8%), were obtained from Sigma-Aldrich (St. Louis, MO, USA), and the internal standard of hydrochlorothiazide (purity 99.5%) was obtained from Institute for Drug Control (Guangzhou, China), while methanol (LC-MS grade) and ammonium acetate were purchased from Sigma-Aldrich.
Firstly, 100 μL of fixed serum was pipetted into 1.5 mL polypropylene tube, and 500 μL hydrochlorothiazide in methanol solution (25 μg/mL) was added to precipitate the protein. The tubes were mixed by vortex for 1 minute. After centrifugation at 12,000 rpm for 10 minutes, 100 μL clear supernatant was extracted and mixed with 100 μL ddH₂O to filter into a new polypropylene tube, and transferred to an auto-sampler. The curve standards were prepared at 0.25, 0.5, l, 5, 10, 50 μg/mL in methanol for IS, dried out using vacuum spinner (Thermo, Waltham, MA, USA), re-dissolved with normal rats' serum, and finally analyzed as described in the serum method. Low, medium and high concentrations of quality-control (QC) samples were also prepared. The IS-quantitative method was described in our previous studies [42 ].