Abstract
On‐line two dimensional liquid chromatography (2D LC) was used to determine apolipoprotein A‐I (apo A‐I) in human plasma without sample pretreatment. The water soluble apolipoproteins were separated from other plasma proteins and lipids by reversed phase (RP) LC in the first dimension using acetonitrile/20 mM aqueous ammonium acetate as mobile phase. A strong anion exchange (SAX) column was subsequently employed in the second dimension to separate the apo A‐I in the apolipoprotein fraction using pH gradient elution with simple buffer mixtures of ammonium acetate and ammonium formate as mobile phase. Compared to other methods requiring separation of high density lipoprotein (HDL) fraction by ultracentrifugation and delipidation of apolipoproteins with organic solvents, loss of apo A‐I is avoided in the on‐line 2D approach. The concentration of apo A‐I in plasma, determined by using the calibration curve for commercial apo A‐I, was found to be 1.12 mg/mL. The identity of apo A‐I isolated from human plasma was confirmed by trypsination and peptide mass fingerprinting (PMF).
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