2.5. Arsenic Speciation Analysis
Arsenic Content, Speciation, and Distribution in Wild Cordyceps sinensis
Evid Based Complement Alternat Med, Feb 19, 2021; DOI: 10.1155/2021/6651498

The extraction methods were performed with reference to the method of Guo et al. [21] and Zhou et al. [23]. Approximately 0.5 g of each powder sample of C. sinensis was added to 10 mL of 0.15 mol/L dilute nitric acid solution and soaked in the mixture overnight. Then, the mixtures were heated in an incubator for 150 min at 90°C and shaken for 1 min every 30 min. The mixtures were cooled to room temperature and centrifuged at 8000 r/min after heat extraction. The supernatants were removed, and 5 mL of 0.15 mol/L dilute nitric acid solution was added to the residue. The extraction was repeated using the procedure described above. The combined supernatants from the two rounds of extraction were analyzed immediately after filtration with a 0.22 μm PTEF membrane. A corresponding reagent blank was made, and the procedure was performed in triplicate.

The different species of arsenic were separated by HPLC and detected by ICP-MS. An anion-exchange column (Hamilton RPR 100 column, 250 mm × 4.6 mm, 5 μm) was used for separation with a gradient system of eluent A, 10 mmol/L NH4H2PO4 (containing 1% methanol V/V, NH3·H2O adjusted pH 9.7), and eluted with B, 40 mmol/L NH4H2PO4 (containing 1% methanol V/V, NH3·H2O adjusted pH 6.7) solution, at a flow rate of 1.0 mL/min. The HPLC elution condition was achieved using the following procedures. For eluent A, 100.0% initial proportion, 100.0% maintained for 4.0 min; linear decrease to 0.0% at 4.5 min and 0.0% maintained for 13.5 min; linear increase to 100.0% at 14.0 min; equilibrium maintained for for 4.0 min. The total sample injection time was 18.0 min, and the acquisition time was 14 min. The injection volume was 10.0 μL.

The method of arsenic speciation analysis was validated by the standard addition method, and the recoveries of each arsenic speciation were used for evaluating the method feasibility. The total arsenic in the extraction solution and the residue of the extracted sample were ingested by microwave digestion method, and the total arsenic was determined by the method in Section 2.4. Then, total arsenic results of extraction solution and residue of extraction were compared with the results of sum of six arsenic species in C. sinensis.