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Protocol

HPLC method for ascorbic acid determination.

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1. Preparation of blood samples. 1) Spin 1 ml blood to separate plasma from blood cells at 2000 rpm. 2) Precipitate 200 µl of plasma with 800 µl of ice cold 90 % methanol containing 1 mM EDTA 3) Incubate plasma sample for 10 min on ice 4) Centrifuge the sample at 12000 rpm for 10min. 5) Save supernatant, and store the supernatant at -700C until needed.
2. HPLC measurement. Column; mBondapakTM C18 Radial-PakTM cartridge (8 X 100 mm). Mobile phase; 100 mM Na-P (pH 4.8) containing 35 % methanol. Flow rate; 1 ml/min Detection; Coularray or an electrochemical detector OR Column: Beckman: C18 column Mobile phase: Na-P (13.7 g/2L), Na-Acetate (8.2g/2L), Dodecyltrimethylamine Chloride (100 mg/2L), and Methanol (600 mL) containing tetraocthylamonium bromide (40 mg); Adjust pH 4.8 with phosphoric acid. Flow rate; 1 ml/min Detection: Coularray or an electrochemical detector Procedure 1. Prepare standards in mobile phase. 2. Run standards in HPLC. 3. Construct a standard curve. 4. Run samples in HPLC. 5. Determine concentrations of samples.
HPLC method for ascorbic acid determination.

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