Development of a method for quantitation of retinol and retinyl palmitate in human serum using high-performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

Abstract

A method for the quantitative analysis of the vitamin A compounds all-trans-retinol and all-trans-retinyl palmitate was developed using high-performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (APCI–LC–MS). Unlike previous quantitative mass spectrometric methods for vitamin A, HPLC separations were carried out using a C30 reversed-phase column instead of GC separation. Because no sample hydrolysis or derivatization was necessary, retinyl palmitate was preserved for analysis instead of being hydrolyzed to retinol. Human serum was analyzed following simple hexane extraction without saponification or any additional purification. A comparison of APCI and electrospray ionization showed that only APCI produced a linear response over all four orders of magnitude of retinol and three orders of magnitude of retinyl palmitate concentrations. Selected ion monitoring of the fragment ion of m/z 269 was used for APCI quantitation of both retinol and retinyl palmitate, since it was the base peak and the only abundant ion in the mass spectra of both compounds and the internal standard, retinyl acetate. The ion of m/z 269 corresponded to loss of water, loss of palmitic acid, or elimination of acetic acid from the protonated molecules of retinol, retinyl palmitate and retinyl acetate, respectively. The limit of detection of APCI–LC–MS for all-trans-retinol and all-trans-retinyl palmitate was determined to be approximately 34 fmol/μl and 36 fmol/μl (0.670 pmol all-trans-retinol and 0.720 pmol all-trans-retinyl palmitate injected in 20 μl on-column), respectively. The limit of quantitation was approximately 500 fmol/μl and 250 fmol/μl (10 pmol and 5 pmol injected in 20 μl on-column) for retinol and retinyl palmitate, respectively.