Solid-phase extraction of phospholipids from hemoglobin solutions using Empore styrene–divinylbenzene disks

A Simple and Improved Method for Extraction of Phospholipids from Hemoglobin Solutions

“…Since hemoglobin solutions made for manufacturing HBOC products may contain trace amounts of contaminated phospholipids, a significant amount of the lipids may be protein-associated. In fact, it has been reported that about 15% of phospholipids spiked in a hemoglobin solution was not extractable by a solid-phase extraction method [12], presumably due to association of the lipids with hemoglobin. Such a "phospholipid masking effect" of Hb solutionn would result in underestimating of the phospholipid concentrations in HBOC products and constitute a significant concern in analysis of trace amounts of contaminated phospholipid in Hb solutions from a product safety point of view.…”

“…. 12), the physical state of precipitated hemoglobin underwent significant changes from tight packed clumps with significant pigment in the solution, to loosely packed clumps with slight pigment in the solution, to finely divided particles in a colorless solution, and then to an even more finely divided flocculent form without color in the solution (Table 2). However, at a lower temperature, such as on ice, a small proportion of isopropanol would result in finely divided particles of precipitated hemoglobin with no pigment contamination in the solution (data not shown).…”

“…For this purpose, various kinds of lipid extraction techniques, both in liquid-liquid format [10][11] and in solid format [12], have been tested or developed. In liquid-liquid extraction format, several widely used methods for extraction of lipids, such as the Folch's method [13] or its modified version described by Bligh and Dyer [14], have been tested for use in extraction of lipids from erythrocytes [10] and hemoglobin solutions [11].…”

“…Since Hb or HBOC solutions are made through a very expensive process, it is desirable to consume as less amounts of samples as possible in QC/QA process. In order to avoid these disadvantages, a solid-phase extraction system using styrene-divinylbenzene (SDB) macroreticular resins has been applied in extraction of phospholipids from erythrocytes and hemoglobin solutions [12]. This method is relatively simple and requires relatively less organic solvents.…”

Stroma-Free Hemoglobin from Bovine Blood

“…For the extraction of phospholipids from red blood cells or aqueous solutions of hemoglobin, styrene-divinylbenzene commercial disks were used. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and sphingomyelin were recovered at 92% yield in average, whereas the recovery yield of phosphatidylserine was 65% [20]. More recently, bovine hemoglobin (HbBv) was purified by a two-step process [21].…”