Quantitative analysis of phospholipids from whey protein concentrates by high-performance liquid chromatography with a narrow-bore column and an evaporative light-scattering detector

A procedure for separation and quantitative determination of phospholipid classes by high-performance liquid chromatography with a narrow-bore column and a light-scattering detector was developed. Cerebrosides (CER), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomeyelin (SPH), and lysophosphatidylcholine (LPC) were base-line resolved, and column life was improved due to low back pressure and low alkalinity of the solvents. Solvent consumption was reduced by 80%, and the detection limit was improved more than tenfold, compared to an analytical column. A gradient elution with pH modifier was necessary for good resolution of acidic phospholipids. A binary solvent system, consisting of A: chloroform/methanol, 80:20 and B: chloroform/methanol/water/ammonium hydroxide (20%), 60:34:6:0.25, was used. Analysis was completed in 36 min, and repeated injections of the samples were possible. The method was applied to the analysis of phospholipids from whey protein concentrates (WPC). Phospholipids in WPC (75% protein) contained (%, w/w) 3.57±0.13 CER; 18.13±1.23 Pl; 4.45±0.21 PE; 7.45±0.58 PS; 30.54±1.84 PC; 35.82±1.16 SPH; and no detectable LPC.