Abstract: To determine glycerol-3-phosphate (I, glycerol 1-phosphate), sample (30 µL) was injected into a stream (0.7 ml/min) of 0.1 M succinate buffer of pH 6 and passed through a column (3 cm x 2.5 mm i.d.) at 30°C containing glycerol-3-phosphate oxidase immobilized on controlled pore glass. The eluate merged with a stream (0.7 ml/min) of 0.1 M carbonate buffer of pH 10.5 containing 10 µM-luminol and 10 µM-Co(II), and after travelling 2.2 cm the flow passed through a glass coil (10 cm x 1 mm i.d.) in front of a photomultiplier tube where the chemiluminescence was measured. To determine glycerophosphorylcholine (II; α-glycerophosphorylcholine) offline conversion to I using phospholipase-D was carried out in 0.1 M succinate buffer of pH 6 at 30°C for 10 min. The detection limits were 0.5 µM-I and 1 µM-II and the RSD were M and 20-100 µM, respectively. Sample throughput was 40/h. Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5 x 10^-7 M and 1 x 10^-6 M respectively with RSD < 2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months.

Abstract: A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically. Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute. The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic-spectrophotometric procedure. An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.