Serum samples were allowed to equilibrate to room temperature. For analysis, 50 μL IS-WS was gravimetrically added to the serum to get an approximately 1:1 mass ratio of analyte to IS. Then 1 mL methanol was slowly added to the sample blends to precipitate protein, while mixing gently on a vortex mixer for 30 min. The sample blends were then centrifuged at 13,000 rpm for 10 min. The resulting supernatant was transferred into a 5-mL centrifuge tube which was added 500 μL water initially, then vortexed for 10 min. The above mixture was transferred to Waters Oasis MAX 1 cc (30 mg) extraction cartridges which were preconditioned with methanol (1 mL) followed by water (1 mL). The loaded cartridges were washed sequentially with water containing 5% ammonium hydroxide (1 mL) and methanol (1 mL). The GCA was eluted from the cartridge with 1 mL methanol containing 2% formic acid. The eluate was dried under nitrogen at room temperature and reconstituted with 200 μL 25% acetonitrile/water (v/v).
Fifty microliters of IS-WS was added into gravimetrically standard working solution to obtain mass ratios of GCA to the IS of 0.8 and 1.2, respectively. The working solution blends were pretreated as the above sample pretreatment.
Fifty microliters of IS-WS was added into gravimetrically standard working solution to obtain mass ratios of GCA to the IS of 0.8 and 1.2, respectively. The working solution blends were pretreated as the above sample pretreatment.