Studies on phospholipids. 4. Determination of ethanolamine and serine

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Abstract
A method depending on a differential color reaction for the determination of ethanolamine and serine in phospholipids is described. A quantity of dinitrophenylated lipid which corresponds to between 0.1 and 1.5 umoles of amino N is dissolved in 5 ml of ethanolic [image]HCl and hydrolyzed by boiling under reflux for 3 hours. The hydrolysate is evaporated to dryness in vacuo, 2 ml of [image] HCl is added and the mixture is again re-fluxed for a further 3 hours. After adding about 0.1 g of Hyflo Supercel the mixture is filtered, the filter is washed with 5 ml of water and the filtrate is reduced to dryness by heating in vacuo. The residue is dissolved in 10 ml of a mixture of dimethylformamide-ethanol-n-butylamine (4:1:0.04, by volume),-0.1 ml of 25% (w/v) tetramethylammonium hydroxide is added and, after mixing, the extinctions are measured at 393 and 500 mu. If e and s respectively represent the amounts of DNP-ethanolamine and DNP-serine in moles in the 10.1 ml of solution and if E1 and E2 represent the extinctions respectively at 393 mu and at 500 mu then it follows that E1 = ae/1.01 + bs/1.01 and E2 = ce/1.01 + ds/1.01. The constants a, b, c and d are equal to 10-4 times the molecular-extinction coefficients; the mean values and standard errors (12 determinations) are: a, 1.13 [plus or minus]0.007; b. 1.13 [plus or minus]0.007; c. 1.23 [plus or minus] 0.007 and d, 0.184 [plus or minus] 0.006. Hence: e = 0.965E2 - 0.157E1; s = 1.05E1 0.965E2; e + s = 0.894E1; and e/(e + s) = 1.18 = 1.08E2/E1.