Hplc grade hexane

Astaxanthin standards (CAS No. 472-61-7, 98.1%), LC–MS-grade formic acid (CAS No. 64-18-6, ≥96%), methanol (CAS No. 67-56-1, ≥99.9%), and LC–MS-grade water were purchased from (TMRM) Tan-Mo Technology Corporation (Changzhou, China). Lutein (CAS No. 127-40-2, 92.6%), fucoxanthin (CAS No. 7176-02-5, 99%), β-carotene (CAS No. 7235-40-7, 99.9%), l-sepiapterin (CAS No. 17094-01-8, 95%), and LC–MS-grade acetonitrile (CAS No. 75-05-8, ≥99.9%) were purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China). Analytical reagent petroleum ether (CAS No. 8032-32-4) and HPLC-grade hexane (CAS No. 110-54-3, ≥95%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). A nylon filter membrane with a 0.22 µm pore size was purchased from Tianjin JINTENG Experimental Equipment. Co., Ltd. (Tianjin, China). The l-sepiapterin standard was dissolved in methanol, and standards for other pigments were dissolved in acetonitrile. The gradient concentrations of all standard working solutions were 31.25, 62.5, 125, 250, 500, and 1000 ng/mL. Methyl tert-butyl ether (MTBE) (CAS No. 1634-04-4) and NH₄OH (CAS No. 1336-21-6) were purchased from Chengdu Chron Chemicals Co., Ltd. (Chengdu, China).

Measurement of β-asarone in brain tissues was performed by High Performance Liquid Chromatography (HPLC) analysis. 1g of brain tissues were grinded in mortar with 1ml of cold phosphate-buffered saline (pH 7.4). The brain tissue was transferred into a tube to homogenize by sonication (50W×15s) on ice. β-asarone was extracted by 1.5ml HPLC-grade hexane (Sinopharm Chemical Reagent Co., Ltd), and then the organic layer was filtered. β-asarone was separated under isocratic condition using 70% HPLC-grade methanol (Sinopharm Chemical Reagent Co., Ltd) in ultrapure water and a column (Inertsil ODS-3, 5μm, 4×250mm). The absorbance of the organic layer was measured at 257nm. Compound identification and analysis calibration were based on use of β-asarone as external standards.