All-trans Retinyl palmitate

Fortified fluid milk (whole, semiskimmed, skimmed) Mix 2 ml milk and 5 ml absolute EtOH in a centrifuge tube, let stand 5 min. Vortex-mix with 5 ml hexane, let stand 2 min. Repeat mixing and standing procedure twice. Add 3 ml water, mix by inversion, centrifuge. LiChrosorb Si-60 5 pm 250 X 3.2 mm Hexane/diethyl ether, 98 2 All-trans-retinyl palmitate UV 325 nm 83... 

Chromatograph the System Suitability Preparation, and measure the peak areas as directed under Procedure. The resolution, R, between the all-trans retinyl acetate and the all-trans retinyl palmitate peaks is not less than 10 (see System Suitability in High-Performance Liquid Chromatography under Chromatography, Appendix IIA), and the relative standard deviation for replicate injections is not more than 3.0%. 

Procedure Separately inject equal volumes (about 40 pL) of the Standard Preparation and the Assay Preparation into the chromatograph, record the chromatograms, and measure the peak areas for the all-trans retinyl acetate (or palmitate) and the 13-cis retinyl acetate (or palmitate), if present, obtained from the Standard Preparation and the peak areas for the all-trans retinyl acetate (or palmitate) and the 13-cis retinyl acetate (or palmitate), if present, in the chromatogram of the Assay Preparation. The relative retention times are about 0.7 for 13-cis retinyl acetate and 1.0 for all -trans retinyl acetate or the relative retention times are about 0.8 for 13-cis retinyl palmitate and 1.0 for all -trans retinyl palmitate. 

Typical chromatograms obtained by high-performance liquid chromatography (HPLC) of the retinyl esters from human RPE are illustrated in Fig. 5. When conditions are optimized for isomer separation (normal phase, uj r half of Fig. 5), there are two major peaks (2, 4), each with a shoulder on the leading edge. Peak 2 is 11-cw-retinyl palmitate the shoulder corresponds to 11-cij-retinyl stearate. Peak 4 is all-trans-retinyl palmitate the shoulder corresponds to all-trans retinyl stearate. Peak 5 is all-rra/u-retinyl stearate, and peak 3 (which is sometimes composite) is unidentified at present. Peaks 2 and 4 were collected separately and injected onto a reverse-phase column. Under these conditions the two component esters were completely resolved, as shown in the lower half of Fig. 5 (peaks 2.1, 2.2 and 4.1, 4.2). 

A slow appearance of labeled 11-c/s-retinyl palmitate in the RPE is demonstrable after injection of all-fran5-[ll,12- H]retinol directly into the eyes of dark-adapted frogs (Fong et al., 1983a). Initially, the all-frans-retinol is taken up by the RPE and converted to all-trans-retinyl palmitate. After several hours, the all-fra i-retinyl palmitate is found to have a fairly constant specific activity, as shown in Fig. 15. No labeled 1 l-cis isomer is detectable at first, but between 2 and 7 h, radioactivity starts appearing in the 11-cis-retinyl palmitate. After 15 h, however, Fig. 15 shows that its specific activity is still only 20% of that of the... 

Fig. S. Retinyl esters stored in human RPE. Analysis by high-performance liquid chromatography (HPLC). (A) Normal-phase column peak 2, ll-c -retinyl palmitate and stearate (shoulder) peak 3, unidentified peak 4, all-rraru-retinyl plamitate and stearate (shoulder) peak 5, ail-trans-retinyl oleate. (B) Peaks 2 and 4 from the normal-phase HPLC eluent were collected and analyzed spearately in a reverse-phase system. The standards consisted of 11-cis-retinyl palmitate (P) and stearate (S). The 11 -cis and all-trans isomers have the same retention time in this system. Peak 2.1, 11-cis-retinyl palmitate peak 2.2, 1 l-cis-retinyl stearate peak 4.1, all-rrons-retinyl palmitate peak 4.2, all-traru retinyl stearate. (From Bridges et al., 1982.)...

Figure 7 Positive ion APCI mass spectra recorded during the LC-MS analysis shown in Figure 6 including (A) aU-fran -retinol at 16.1 min, (B) all-tran -retinyl acetate at 21.9 min, and (C) all-franj-retinyl palmitate at a retention time of 38.5 min. Note that all three retinoids fragmented during APCI to form a common base peak at m/z 269, which was used for selected ion monitoring during quantitative analysis. (From Ref. 310.)...

Retinyl palmitate [79-81-2] M 524.9, e m (all-trans) 1000 (325 nm) in EtOH. Separate from retinol by column chromatography on water-deactivated alumina with hexane containing a very small percentage of acetone. Also chromatographed on TLC silica gel G, using pet ether/isopropyl ether/acetic acid/water (180 20 2 5) or pet ether/acetonitrile/acetic acid/water (190 10 1 15) to develop the chromatogram. Then recrystd from propylene. 

Of the vitamin A commonly found in foods, only all-tran.v-retinol and smaller amounts of 13-ds-retinol, both in esterified form, are usually present in significant quantities. For the analysis of vitamin A-fortified foods, HPLC can be applied to determine either the total retinol content or the added retinyl ester (acetate or palmitate), depending on the extraction technique employed. 

Fig. 1. The structures of key retinoids and their precursors. Fish convert retinyl esters (e.g. retinyl palmitate (RP)) and carotenoids (e.g. /3-carotene) to retinol in the gut lumen prior to intestinal absorption. Retinyl esters (e.g. RP) stored in the liver are synthesized from retinol by lecithin retinol acyltransferase (LRAT) and acyl CoAiretinol acyltransferase (ARAT). The retinyl esters are mobilized through their conversion to retinol by retinyl ester hydrolase (REH), which is then transported in the circulation to various sites in the body. Retinol is further metabolized within specific tissues to retinal by alcohol dehydrogenases (ADH) or short-chain dehydrogenase/reductase. Retinal is converted to the two major biologically active forms of retinoic acid (RA) (all-trans and 9-cis RA). Retinaldehyde dehydrogenase 2 (Raldh2) synthesizes all-trans RA from all-trans precursors and 9-cis RA form 9-cis precursors.

Fig. 2. HPLC analysis of neutral retinoids in tissues and plasma Samples were eluted from a reverse-phase Waters ODS column (1.2 x 10 cm) at 2 mL/min with a linear gradient of 15% water in methanol to methanol over 20 min, followed by 30 min of methanol. 1, 5,6-epoxyretinol 2, aU-trans-retinol, 3, all-/ra -retinal, 4, anhydroretinol, 5, retinyl docosahexanoate 6, retinyl palmitoleate 7, retinyl linoleate 8, retinyl palmitate and retinyl oleate, 9, retinyl stearate These specific examples illustrate the analyses of neutral-tissue retinoids equilibrated with orally fed [ HJretinol and extracted from (A) rat small-intestine mucosa (dashed line) or kidney (solid line) (B) plasma (dashed line) or liver (solid line) The concentrations of retinol and most retinyl esters in tissues and blood, however, are sufficiently large for detection by UV at 325 nm.

Collins M, Tzimas G, Hummler H, Buergin H, Nau H (1994) Comparative teratology and transplacental pharmacokinetics of all-tran -retinoic acid, 13-cw-retinoic acid and retinyl palmitate following daily administration in rats. Toxicol Appl Pharmacol 127 132-144... 

---