Retinoids, separation

Retinal is not always well resolved from retinol in reversed-phase HPLC systems resolution is affected both by the stationary phase and by the mobile phase composition. Curley et al. found that methanol water was superior to acetonitrile water for resolution of retinol from retinal on an end-capped C18 column, whereas acetonitrile water was superior for separating retinal from retinol on a non-end-capped column (130) this difference presumably is due to hydrogen bonding between the analytes and the mobile phase or stationary phase. Plots of log (k ) versus carbon number are linear and parallel for reversed-phase separation of alcohol versus aldehyde/ketone analogs of apo-retinoids and apo-carot-enoids using methanol water mobile phases, but nonparallel for acetonitrile water mobile phases (H.C. Furr, unpublished observations). Thus, separation of retinol and retinal is more dependent on the specific column and mobile phase than are most retinoid separations. 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

Ethanol also inhibits ADH-catalyzed retinol oxidation in vitro, and ethanol treatment of mouse embtyos has been demonstrated to reduce endogenous RA levels. The inhibition of cytosolic RolDH activity and stimulation of microsomal RolDH activity could explain ethanol-mediated vitamin A depletion, separate from ADH isoenzymes. Although the exact mechanism of inhibition of retinoid metabolism by ethanol is unclear, these observations are consistent with the finding that patients with alcoholic liver disease have depletedhepatic vitamin A reserves [review see [2]. 

Kagechika H (2002) Novel synthetic retinoids and separation of the pleiotrophc retinoidal activities. Curr Med Chem 9(5) 591-608... 

Gundersen and Blomhoff (1999) used online dilution with online SPE to measure vitamin A (retinol) and other active retinoids in animal plasma. The intention of online dilution in this application was on optimizing SPE extraction conditions rather than on peak focusing during analytical separation. An SPE cartridge packed with Bondapak C18 materials (37 to 53 jt/M, 300 A, Waters, Milford, Massachusetts) and a reversed-phase analytical column (250 x 2.1 mm inner diameter, Superlex pkb-100, Supelco, Bellefonte, Pennsylvania) were controlled by a six-port switching valve (Rheodyne, Cotati,... 

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

Fig. 2.37. Gradient LC separation of the retinoid solution components and retinoic acid isomers by (A) UV-DAD detection (350 nm) and (B) fluorescence detection with on-line photoreactor switched (a) off and (b) on with irradiation at 366 nm. Peak identification 1 = 13-civ retinoic acid 2 = 9-civ retinoic acid 3 = all-fraws retinoic acid 4 = vitamin A palmitate 5 = /1-carotene. Reprinted with permission from R. Gatti el al. [85].

Retinoids The challenge in fat-soluble vitamins analysis is to separate them from the lipid fraction that contains interferents. Alkaline hydrolysis, followed by LLE, is widely applied to remove triglycerides. This technique converts the vitamin A ester to all-trani-retinol. A milder process, which does not hydrolyze vitamin A ester, is alcoholysis carried out with metha-nolic KOH solution under specific conditions that favor alcoholysis rather than saponification. A more accurate explanation of this technique is reported in the book Food Analysis by FIPLC [409]. For some kind of matrices a simple liquid extraction can be sufficient with [421-423] or without [424,425] the purification... 

Goodwin has provided a useful summary of the structural chemistry applicable to the retinoids of vision36. However, the book is not as comprehensive as needed to address all of the types of Vitamin A. He also refers to a separate essay on the structural notation he uses37. Taylor Ikawa have provided additional nomenclature, more semisystematic names and excellent information on laboratory evaluation techniques38. 

The above distinctions require a clear separation between the passive substrate material (even though it contains a retinoid) and the photon sensitive chromophores of vision (which contain a different family of retinoids). For the... 

With hydroxylamine, al -trans, ll-c/5-, and 13-cw-retinal gave a mixture of the syn- and anti-oximes, whereas 11,13-di-cw-retinal gave only the syn-oxime/ The synthesis of the dansyl-lysyl-lysine-N-retinylidene Schiff base has been described/ The products of a colour reaction of retinoic acid (121) in 74% H2SO4 have been identified as (122) and (123)/ The oxidation and isomerization of retinoic acid by I2 and light have been used to prepare the a -trans- and 13-CI5-isomers of 4-oxoretinoic acid (124) which were separated by h.p.l.c/ The photoisomerization of the retinoid (125) has been studied. The many isomers produced were separated by h.p.l.c. and characterized by H and n.m.r. ... 

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