The analyte fingolimod and its internal standard, findgolimod-d4, were extracted using an automated liquid-liquid extraction, and the extracted samples were loaded onto an ACE 3 columnlumn (50 mm × 4.6 mm, 3 μm, Life Science, Peterborough, Canada) for separation.
During the pretreatment procedure, the target compounds were extracted from the samples using accelerated solvent extraction (ASE), and the extracted samples were then purified using four steps for clean-up: liquid liquid extraction (LLE), Florisil clean-up, aqueous alkali extraction (AAE) and hydrophilic lipophilic balance (HLB) enrichment.
The extraction was repeated (x2), and the extracted samples from each pad were pooled and then purified using C18 columns (The Nest Group Inc., Southboro, MA) before running MS. OGE of peptides was carried out using 24-cm IPG strips, pH 3-10 (GE Heandhcane), and an OGE fractionator [ 17] (Agilent Technologies, Inc). according to the instructions accompanying the equipment.
SPVs of the extracted samples are generally larger than that of the un-extracted ones.
The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions.
Scanning electron microscopic analysis of the extracted samples was performed to identify the types of deposits.
The analyte responses were compared and the statistical results revealed the feasibility of direct analysis of the extracted samples in carbon tetrachloride, resulting in a convenient methodology.
The HPLC analysis of the extracted samples was used in conjunction with response surface design to optimize four operating variables of supercritical CO2 extraction (pressure, temperature, CO2 flow rate and extraction dynamic time).
By sequencing the addition of veratridine and ouabain, with either the extracted samples, saxitoxin, tetrodotoxin, or ciguatoxin (CTX-a sodium channel activator) to the RBCs a sodium channel antagonist or activator can be detected.
