Retinoids method

Rockley, N. L Rockley, M G, Halley, B. A., and Nelson, E. C (1986) Fourier transform infrared spectroscopy of retinoids Methods Enzymol 123,92-101. 

Lotan, R, Lotan, D, and Sacks, P G. (1990) Inhibition of tumor cell growth by retinoids. Methods Enzyntol 190, 100-110... 

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). 

Breithaupt, D.E. and Schwack, W., Determination of free and bound carotenoids in paprika Capsicum annuum L.) by LC/MS, Eur. Food Res. TechnoL, 211, 52, 2000. Epler, K.S., Ziegler, R.G., and Craft, N.E., Liquid chromatographic method for the determination of carotenoids, retinoids and tocopherols in human serum and in food, J. Chrvmatogr, 619, 37, 1993. 

A retinoid analog was synthesized using the cross-coupling method (Equation (192)). 

An online filter was also used to protect the analytical column. A guard column was used before the T to prevent breakthrough. A restrictor was used to balance the pressure before, during, and after the valve switches. After sample transfer, the valve was switched back. With this method, both water-insoluble retinoids and water-soluble retinoic acid were extracted simultaneously. Because of the minimal light exposure of these light-sensitive analytes during the procedure, an extraction recovery of 97 to 100% was achieved with a quantitation range of 100 fmol to 3 nmol. 

The ability to form carbon—carbon bonds in a controlled manner around an alkene is the subject of continuing intense research [49,134—136], These compounds are stable and, due to the considerably different reactivities of the C—Zr and C—B bonds, allow for selective and sequential reactions with a variety of electrophiles. Temarotene 58 is a retinoid of interest [137] because it shows no sign of hypervitaminosis A and it is not teratogenic, presumably due to the lack of a polar group [138,139], The published synthesis of temarotene-type compounds is long and leads to mixtures of diastereo-isomers, from which the desired product is eventually isolated [140—142], However, the synthesis of temarotene 58 by the method of Srebnik et al. [130] is straightforward, as outlined in Scheme 7.18. 

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],... 

Another general method for the preparation of 19,19,19- and 20,20,20-trifluor-oretinals involves the aldol condensation of trifluoroacetone with an ethylenic aldehyde. When performed in the presence of acetic acid and piperidine, it affords an a, S-unsaturated ketone. Applied to cyclocitral and its homologues, the method leads to polyenic trifluoromethyl ketones. From these ketones, the various tri-fluorinated retinoids are prepared by known methods (Figure 4.24). ... 

Frolick Olson describe the conventional methods of extracting retinoids from biological samples236. They stress the delicacy of the retinoids and describe two important points to consider, (1) whether the method chosen will result in complete extraction of the retinoid of interest, and (2) whether it will contribute to the production of artifacts. Unfortunately, they do not consider whether the product itself could be degraded. They do stress the sensitivity of these labile compounds to both hydrolysis and oxidation. They also address the use of fluorescence in assays. This work points out that the use of fluorescence is a negative test for the retinoids of vision when in liquid crystalline form. They point out that mass spectrometry is one of the most specific forms of assay for the retinoids. The utility of this technique will be discussed in Chapter 6. They do not describe how the techniques mentioned are able to remove a retinoid from within the putative rhodopsin molecule, or how to do in without damage to the retinoid. 

Of the two primary methods of isolating the retinoids, saponification and direct extraction, saponification is almost bound to destroy any actual resonant retinoids present. Direct extraction promises more success. However, even in this method, care must be taken to avoid oxidation, or other modifications (including rearrangements) of the chromophores. Roberts, et. al.238 has shown that significant levels of hydrolysis occurred in both the direct extraction method, using the sodium sulfate procedure of Ames et. al., and the lyophilization method of Ito. It appears that hydrolysis leads to retinol as an end product. This accounts for the common assumption that retinol (which was found in the final residue) must have been the original chromophore (with or without an associated opsin). 

When attempting to extract and characterize the retinoids of vision, it is best to consider the above factors and then to follow Spom, Roberts Goodman s advice239 In conclusion, before any extraction procedure is applied to the removal of retinoids from tissue samples, it is essential that the method be thoroughly examined with the appropriate controls. Without this close examination, of methods used, the data obtained could be both difficult to reproduce and unrelated to physiological events. More specifically, the data will probably only apply to the residue, retinol, resulting from excessively aggressive extraction techniques. At best, it will apply to the Rhodonines in dilute solution. 

Much of the new chemistry on the coupling of alkynyl halides has dealt mostly with applications of known methods. The Cadiot-Chodkiewicz-type coupling reactions (i.e. terminal acetylene coupling with alkynyl halides) have formed the cornerstone of synthesis of acetylenic retinoids (i.e. 9)58. 

Epler, K.S. Ziegler, R.G. Craft, N.E. 1993. Liquid chromatographic method for the determination of carotenoids, retinoids and tocopherols in human serum and in food. J. Chromatogr. Biomed. 619 37-48. 

Bonchev, D., Mountain, C.F., Seitz, W.A. and Balaban, A.T. (1993c). Modeling the Anticancer Action of Some Retinoid Compounds by Making Use of the OASIS Method. J.Med.Chem., 36,1562-1569. 

Retinoic acid, an endogenous retinoid, is a potent inducer of cellular differentiation. Because cancer is fundamentally a loss of cellular differentiation, circulating levels of retinoic acid could play an important role in chemoprevention. However, physiological concentrations are typically below the limits of HPLC detection. Sensitive techniques, such as negative chemical ionization (NCI) GC/MS have been employed for quantification, but cause isomerization and also fail to resolve the cis and trans isomers of retinoic acid. Normal phase HPLC can resolve the cis and trans isomers of retinoic acid without isomerization, and mobile phase volatility makes it readily compatible with the mass spectrometer. Based on these considerations, a method combining microbore normal phase HPLC separation with NCI-MS detection was developed to quantify endogenous 13-cis and all-trans retinoic acid in human plasma. The limit of detection was 0.5 ng/ml, injecting only 8 pg of retinoic acid onto the column. The concentration of 13-cis retinoic acid in normal, fasted, human plasma (n=13) was 1.6 +/- 0.40 ng/ml. 

Introduction.—This Report covers the literature published up to approximately the end of September, 1981. Few new carotenoid structures have been reported. The main advances in carotenoid chemistry have been in the stereospecific synthesis of carotenoids with chiral end-groups. Current interest in the possible use of retinoids in cancer chemotherapy has prompted the preparation of a considerable number of retinoic acid analogues. There has been no major new development in the use of physical methods but h.p.l.c. becomes more and more the method of choice for carotenoid separation, purification, and assay, and the increasing number of papers on resonance Raman spectroscopy emphasizes the potential value of this technique in the carotenoid field. 

Physical Methods.—Separation and Assay. A range of isomers of astaxanthin (8) diacetate (9-cis, 13-cis, 15-cis, 9,9 -di-cis, 9,13-di-cw, 9,13 -di-cw, 13,13 -di-cw, 13,15-di-cw), prepared by thermal and iodine-catalysed isomerization of irans-(S) have been separated by h.p.l.c.126 A procedure has been developed for separation of bean leaf etioplast pigments, including carotenoids,127 by h.p.l.c. H.p.l.c. separations of esters of all-trans-, 9-cis-, 11-cw-, and 13-cw-retinol,128-130 and determinations of retinol in serum,131 retinol and 13-cw-retinoic acid,132 and the aromatic retinoid (195)133 in plasma have been described. A reversed-phase ion-pair... 

---