Then, the extract was divided into two 1.5 mL new tubes. Next, 500 mL of chloroform:isopropanol (3:2) was added each tubes and were centrifuged for 5 min at 12,000 rpm. The lower phase was
transferred into 2 mL centrifuge tubes as the first extraction. The remaining methanolic solutions were used for the second extraction by adding 500 mL of chloroform. Then, the lower phases were taken to the tubes. It was called as second extraction. Afterward, Na2SO4 was added to the tubes and mixed by hand and centrifuged at 12,000 rpm for 1 min in order to remove water. Extractions were evaporated to dryness under gentle stream of nitrogen and finally the volume was completed to 0.5 mL with HPLC grade methanol and passed through a PTFE millipore membrane filter (0.22 mm).
Then, the extract was divided into two 1.5 mL new tubes. Next, 500 mL of chloroform:isopropanol (3:2) was added each tubes and were centrifuged for 5 min at 12,000 rpm. The lower phase wastransferred into 2 mL centrifuge tubes as the first extraction. The remaining methanolic solutions were used for the second extraction by adding 500 mL of chloroform. Then, the lower phases were taken to the tubes. It was called as second extraction. Afterward, Na2SO4 was added to the tubes and mixed by hand and centrifuged at 12,000 rpm for 1 min in order to remove water. Extractions were evaporated to dryness under gentle stream of nitrogen and finally the volume was completed to 0.5 mL with HPLC grade methanol and passed through a PTFE millipore membrane filter (0.22 mm).
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