Molybdenum in the determination of phospholipids without acid digestion.

To the Editor: We read with interest a paper on phospholipid analysis in this journal (1) but deferred comment until the publication of our own paper on the subject, which we believe contains relevant material (2). Sandhu refers to the formation of a Prussian blue complex as the result of a reaction between phospholipids and a chromogenic reagent containing molybdenum. Clearly this cannot be so, as Prussian blue is the color produced by the ferroferricyanide ion (Fe(III) [Fe(H)(CN)6]). In a previous publication (3) a similar procedure was described and the spectrum of the resulting color complex shown. This spectrum is identical with that of molybdenum blue, which is the color that would be expected from Sandhu’s method. Molybdenum blue is a colloidal solution of a complex, the exact structure of which is unknown, but which contains Mo(V) and Mo(VI). Because molybdenum blue is the end-point color in many phosphate analyses it is often assumed that phosphate is essential to its formation. This is not so; it merely has a high affinity for Mo(VI). We therefore suggest that in the reaction described by Sandhu some of the molybdate is reduced to Mo(V) by metallic mercury and that his chromogenic reagent is molybdenum blue. This is borne out by its color. We further suggest that when this reagent reacts with phospholipids, a further complex occurs involving either the lipid phosphorus or quaternary nitrogen. Because of the lipophilic nature of the lipid molecule, this complex is transferred to the organic phase in the manner we described (2). In our method, complexing occurs through the quaternary nitrogen, whereas in Sandhu’s method it would appear that the phosphorus is involved. In a similar procedure, Raheja et al. (3) infer that they obtained a positive reaction with phosphatidic acid, which would not be the case if a quaternary nitrogen was involved.