Retinyl palmitate chromatography

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. 

WO Landen Jr, RR Eitenmiller. Application of gel permeation chromatography and nonaqueous reverse phase chromatography to high pressure liquid chromatographic determination of retinyl palmitate and /3-carotene in oil and margarine. J Assoc Off Anal Chem 62 283-289, 1979. 

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%. 

Finally, a rather unusual technique was developed in Japan (25) for the identification of several drugs in pharmaceutical preparations. The technique is called thin-layer slick chromatography (TLSC) and is described as an advanced version of thin-layer chromatography in a cylindrical form. The adsorbent on the stick is a mixture of silica gel, microciystalline cellulose (proportions, 5 2) and 3% of a binder, hydroxyethylcellulose. One to ten microliters of the sample are spotted on the thin-layer stick 2 cm from the lower end and similarly, standard samples are applied on the reverse side, to ensure precise detection. For the identification of retinyl palmitate or retinyl acetate, pure benzene (200 pi) was used as developer. 

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). 

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.)...

High-pressure gel permeation chromatography (pStyragel columns eluted with dichloromethane or tetrahydrofuran) has been used for preliminary purification of retinyl palmitate in foodstuffs (128,129) but has not gained wide usage. 

RB van Breemen, D Nikolic, X Xu, Y Xiong, M van Lieshout, CE West, AB Schilling. Development of a method for quantitation of retinol and retinyl palmitate in human serum using high performance liquid chromatography-ahnospheric pressure chemical ionization mass spectrometry. J Chromatogr A 794 245-251, 1998. R Andreoli, M Careri, P Manini, G Mori, M Musci. HPLC analysis of fat-soluble vitamins on standard and narrow-bore columns with UV, electrochemical and particle beam MS detection Chromatographia 44 605-612, 1997. 

The applications surveyed in Tables 4 through 9 illustrate the general principles of vitamin E assays outlined in I.E. 1. Specifically, each matrix puts a different emphasis on the E vitamers to be determined. Thus, in serum/plasma a-tocoph-erol is clearly the main compound of interest. Accordingly, leversed-phase chromatography with UV detection is the indicated technique for this purpose. In addition, as part of the assessment of the antioxidant status of humans, tocopher-ols are determined concurrently with retinoids (retinol, retinyl palmitate) and carotenoids (particularly p-carotene). a-Tocopherol is also the principal target compound in erythrocytes and platelets but here, predictably, the quantitation of a-tocopherolquinone may also be meaningful as an indicator of oxidative stress (7). The need to assay this minor constituent in turn justifies coulometric detection. The analysis of tissues is complementary to that of plasma and red blood cells and mainly concerns the determination of a-tocopherol as well as retinoids, carotenoids, and ubiquinones (Table 6). 

JL Rudy, F Ibarra, M Zeigler, J Howard, C Argyle. Simultaneous determination of retinol, retinyl palmitate, and a-tocopherol in serum or plasma by reversed-phase high-performance liquid chromatography. LC-GC 7 969-971, 1989. 

Fig. 4. Chromatogram of a mixture of retinyl ester standards. Column, Supelcosil LC-8 (5 jLm) mobile phase, acetonitrile water (88 12) from origin to arrow and acetonitrile water (98 2) for rest of chromatography flow rate, 3 ml/min. The esters of retinol are 1, acetate, 2 0 2, laurate, 12 0 3, 7-linolenate, 18 3 4, myristate, 14 0, 5, palmitoleate, 16 1 6, linoleate, 18 2 7, palmitate, 16 0 8, oleate, 18 1 9, stearate, 18 0 10, arachidonate, 20 4. (Reprinted with permission from Ross, 1981.)...

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