[The correlation between the concentrations of VEGF and PEDF and Ca2+-PKC signaling pathways in human retinal pigment epithelial cells cultured in vitro after exposuring to blue light].

OBJECTIVE: To investigate the concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), inositol triphosphate (IP3) and diacylglycerol (DAG) in human retinal pigment epithelium (RPE) cells after exposuring to blue light, and to explore the relationship with Ca2+-PKC signaling pathways, to evaluate the role of Ca2+-PKC signaling pathways of blue-light irradiation induced apoptosis in RPE cells. METHODS: The fourth generation human RPE cells in vitro were exposured to blue light (2000±500 lux) for 6 hours, 24 hours prolongation of post-exposure culture. The concentrations of VEGF, PEDF, IP3 and DAG were assayed by enzyme linked immunosorbent assay (ELISA). Cells were randomly divided into 6 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+PMA), group D (exposure to blue light+Calphostin C), group E (exposure to blue light+Nifedipine), group F (exposure to blue light+Calphostin C+Nifedipine). Flow cytometry was used to detect the apoptosis rate of human RPE cells in A, B and F group. RESULTS: Comparing with group A (584.38±10.66), the concentration of VEGF in group B (700.70±5.88), group C (698.21±6.66) and group E (648.30±4.91) was higher, the difference was statistically significant (P=0.002, 0.002, 0.016). Comparing with group B (700.70±5.88), the concentration of VEGF in Group D (623.87±3.12) and E (648.30±4.91) was lower (P=0.001, 0.002). Comparing with group A (75.96±1.70), the concentration of PEDF in Group B (71.82±1.67) and C (72.43±0.58) was lower (P=0.004, 0.011), but the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group B (71.82±1.67), the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group A (7.70±0.29), the ratio of VEGF to PEDF in Group B (9.85±0.34) and Croup C (9.64±0.02) was higher (P=0.008, 0.027) Comparing with group B, The ratio of VEGF to PEDF in Group D (7.23±0.08) and E (6.92±0.06) was lower (P=0.016, 0.015). Comparing with group A (108.42±0.75, 995.47± 13.61), the concentration of IP3 and DAG in Group B (117.24±1.06, 1070.10±10.07), C (137.12±2.71, 1046.40±7.90), D (139.17±1.40, 1041.13±9.76) and E (149.61±0.77, 1273.14±10.89) was higher, the difference was statistically significant (P=0.003, 0.007, 0.000, 0.000, 0.000, 0.000, 0.000, 0.000). Comparing with group B, the concentration of IP3 in Group C, D and E was higher (P=0.011, 0.000, 0.000). Comparing with group B, the concentration of DAG in Group C and D was lower (P=0.021, 0.007). Comparing with group B, the concentration of DAG in Group E was higher (P=0.000). Comparing with group A (10.27±1.88), the apoptosis rate of RPE cells in Group B(25.07±2.66) and F(19.37±3.23) was higher, the difference was statistically significant (P=0.001, 0.009). Comparing with group B (25.07±2.66), the apoptosis rate of RPE cells in Group F (19.37±3.23) was lower (P=0.038). CONCLUSIONS: (1) After exposuring to blue light, the concentrations of VEGF, IP3 and DAG are increased and the ratio of VEGF to PEDF is also increased and the concentration of PEDF is decreased in human RPE cells. (2) L-Type Calcium Channels and Ca2+-PKC signaling pathways may be regulate the concentrations of VEGF, PEDF, IP3 and DAG in RPE cells after exposuring to blue light by feedback regulation. (3) The application of Calphostin C combined with Nifedipine may be restrain the apoptosis of RPE cells after exposuring to blue light.

Nano-lipoidal carriers of tretinoin with enhanced percutaneous absorption, photostability, biocompatibility and anti-psoriatic activity.

Tretinoin (TRE) is a widely used retinoid for the topical treatment of acne, psoriasis, skin cancer and photoaging. Despite unmatchable efficacy, it is associated with several vexatious side effects like marked skin erythema, peeling and irritation, eventually leading to poor patient compliance. Its photo-instability and high lipophilicity also pose challenges in the development of a suitable topical product. The present study, therefore, aims to develop biocompatible lipid-based nanocarriers of TRE to improve its skin delivery, photostability, biocompatibility and pharmacodynamic efficacy. The TRE-loaded liposomes, ethosomes, solid lipid nanoparticles (SLNs) and nanostructured lipidic carriers (NLCs) were prepared and characterized for micromeritics, surface charge, percent drug efficiency and morphology. Bioadhesive hydrogels of the developed systems were also evaluated for rheological characterization, photostability, ex vivo skin permeation and retention employing porcine skin, and anti-psoriatic activity in mouse tail model. Nanoparticulate carriers (SLNs, NLCs) offered enhanced photostability, skin transport and anti-psoriatic activity vis-à-vis the vesicular carriers (liposomes, ethosomes) and the marketed product. However, all the developed nanocarriers were found to be more biocompatible and effective than the marketed product. These encouraging findings can guide in proper selection of topical carriers among diversity of such available carriers systems.

Improved tretinoin photostability in a topical nanomedicine replacing original liquid suspension with spray-dried powder with no loss of effectiveness.

OBJECTIVE: The use of spray-dried powders containing tretinoin-loaded nanocapsules instead of the original liquid suspension, aimed at the preparation of dermatological nanomedicines with improved photostability, was investigated. METHODS: Powders were prepared using lactose as a drying adjuvant. Hydrogels were prepared using two approaches: dispersing Carbopol Ultrez 10 in an aqueous redispersion of the powder or incorporating the powder in previously formed hydrogels. RESULTS AND DISCUSSION: The photodegradation of tretinoin in hydrogels prepared with the powders showed similar half-life times (around 19.5 h) compared to preparations with the original liquid nanocapsules (20.7 ± 1.4 h), regardless of the preparation approach. In addition, the topical nanomedicines prepared with the spray-dried powders presented a significant improvement in tretinoin photostability compared to the formulation containing the non-encapsulated drug. CONCLUSION: This study verified that the addition of the spray-dried powders containing tretinoin-loaded lipid-core nanocapsules to hydrogels did not influence the photoprotection of the drug compared with the preparation procedure using the original liquid suspension.

Tretinoin-loaded nanocapsules: Preparation, physicochemical characterization, and photostability study.

The aim of this study was to prepare and characterize tretinoin-loaded nanocapsules as well as to evaluate the influence of this nanoencapsulation on tretinoin photostability. Tretinoin-loaded nanocapsules (0.5 mg ml(-1)) were prepared by interfacial deposition of preformed polymer (poly-epsilon-caprolactone) using two different oily phases: capric/caprylic triglycerides and sunflower seed oil. Tretinoin-loaded nanocapsules presented drug content close to the theoretical value, encapsulation efficiencies higher than 99.9%, nanometric mean size with a polydispersity index below 0.25, and pH values between 5.0 and 7.0. Regarding photodegradation studies, tretinoin methanolic solution showed a half-life time around 40 min according to a first order equation, whereas tretinoin nanocapsule suspensions showed a half-life between 85 and 100 min (twofold higher than in methanolic solution) according to a zero order equation. Tretinoin-loaded nanocapsules improved tretinoin photostability, independently on the type of oily phase used in this study, and represent a potential system to be incorporated in topical or systemic dosage forms containing tretinoin.

UVA is the major contributor to the photodegradation of tretinoin and isotretinoin: Implications for development of improved pharmaceutical formulations.

The chemical stability of tretinoin (RA) and isotretinoin (13RA) in ethanol and dermatological cream preparations exposed to solar simulated light (SSL), UVA, and visible light has been studied. Photostability was monitored by an HPLC method that allowed simultaneous analysis of RA and 13RA, thus allowing photodegradation due to isomerization to other retinoids and photolysis to non-retinoid products to be monitored. Both retinoids undergo both isomerization and photolysis following SSL, UVA and visible light exposure but RA is more sensitive to photodegradation than 13RA. Degradation of both retinoids by photolysis is considerably greater in cream formulations than in ethanol and the photodegradation follows second order kinetics. Rate constants and half-lives for degradation of RA and 13RA in ethanol solution and cream preparations subjected to different light sources are reported. The UVA component of SSL is the major contributor to photodegradation. Since UVA penetrates deeply into skin, our results suggest that photodegradation of RA may contribute to the photosensitivity associated with RA therapy. Our studies suggest that development of improved formulations and the use of effective UVA sunscreens may reduce the side effects of RA therapy.

N-phthaloylchitosan-g-mPEG design for all-trans retinoic acid-loaded polymeric micelles.

The amphiphilic grafted copolymer N-phthaloylchitosan-grafted poly(ethylene glycol) methyl ether (PLC-g-mPEG) was synthesized using chitosan with four different degrees of deacetylations (DD) (80, 85, 90 and 95%). All-trans retinoic acid (ATRA) was incorporated into PLC-g-mPEG by dialysis method in an attempt to optimize carriers for ATRA delivery. Morphological investigation by transmission electron microscopy (TEM) showed that the particles had round and uniform shapes. The particle sizes of ATRA incorporated into micelles were about 80-160 nm depending on the initial drug-loaded and %DD of chitosan. Physicochemical properties of ATRA-loaded polymeric micelles were also investigated. It was found that %DD of chitosan, which corresponded to the N-phthaloyl groups in the inner core of the micelles, was a key factor in controlling the incorporation efficiency, stability of the drug-loaded micelles and drug release behavior. As the %DD increased, the incorporation efficiency and ATRA-loaded micelles stability increased. The sustained release profiles were also obtained at high %DD (90 and 95%). When compared to the unprotected ATRA, ATRA loaded in PLC-g-mPEG micelles was efficiently protected from photodegradation. This result suggested that loading of ATRA in micelles improved the chemical stability of ATRA.

Investigation on the photostability of tretinoin in creams.

In this investigation, the photodegradation of some tretinoin cream formulations was evaluated. Several oils were selected to prepare the cream formulations: olive oil, maize oil, castor oil, isopropyl myristate and Miglyol 812. A solubility study showed that tretinoin is best soluble in castor oil (0.60g/100ml), followed by isopropyl myristate, maize oil, Miglyol 812 and olive oil, respectively, 0.35, 0.30, 0.29 and 0.22g/100ml. The photostability of tretinoin in oils is comparable with the photostability of a tretinoin lotion (ethanol/propylene glycol 50/50), castor oil and olive oil giving slightly better results than the other oils. Investigation of the photodegradation of tretinoin in o/w creams, prepared with the same oils as mentioned above, revealed that tretinoin is far more stable in the cream formulations than in the respective oils, however it is not clear whether this is due to the formulation or due to a different irradiation technique. Tretinoin seemed to be most stable in the olive oil cream, followed by the castor oil cream. However microscopic investigation revealed the presence of tretinoin crystals in the olive oil cream, while the other creams were free of it. As a conclusion, one can say that the cream prepared with castor oil seems to be the most suitable one, in terms of solubility of tretinoin and in terms of photostability.

The effect of simulated solar UV irradiation on tretinoin in tretinoin gel microsphere 0.1% and tretinoin gel 0.025%.

Topical tretinoin is highly effective and widely used in the treatment of acne vulgaris. Tretinoin gel microsphere 0.1% (TGM)--alone or in combination with erythromycin-benzoyl peroxide (EBP) or clindamycin-benzoyl peroxide (CBP) topical gels-and tretinoin gel 0.025% (TG)--alone or, combined with EBP-were exposed to simulated solar UV irradiation to determine the degree of tretinoin photodegradation/isomerization. The investigation revealed that 94% and 84% of the initial tretinoin in the TGM formulation remained stable after 2 and 6 hours, respectively, of simulated solar UV irradiation. When combined with EBP topical gel, 89% and 81% of the initial tretinoin remained stable after 2 and 6 hours, respectively, of exposure to simulated solar UV irradiation; 86% and 80% of the tretinoin remained stable after 2 and 6 hours, respectively, when combined with CBP topical gel. In contrast, only 19% and 10% of the tretinoin remained unchanged after 2 and 6 hours, respectively, of simulated solar UV irradiation of TG. Combined with the EBP topical gel, undegraded tretinoin quantities were further reduced to 7% and 0% at 2 and 6 hours, respectively, with TG. These data suggest that the TGM formulation offers marked protection against tretinoin photodegradation compared with TG, even in the presence of a topical gel containing a potent antibiotic or a strong oxidizing agent. Although simulated solar UV irradiation is not entirely reflective of actual conditions, the results appear to be substantial.

Effect of visible light on normal and P23H-3 transgenic rat retinas: characterization of a novel retinoic acid derivative present in the P23H-3 retina.

Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all-trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480-590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration.

Accelerated photostability study of tretinoin and isotretinoin in liposome formulations.

The photodegradation of retinoic acids, tretinoin and isotretinoin, in ethanol and liposomes was studied. The light irradiation was performed according to the conditions suggested by the ICH Guideline for photostability testing by using a Xenon lamp within a wavelength range of 300-800 nm. The photodegradation process was monitored by UV spectrophotometry. In ethanol solution, tretinoin and isotretinoin undergo complete isomerization just within a few seconds of light exposure to give 13-cis and 9-cis isomers, respectively. The 13-cis isomer from tretinoin undergoes in turn a slow isomerization to the same 9-cis isomer. Both retinoic acids incorporated in liposome complexes showed an increased stability in comparison to the ethanol solutions. In particular for tretinoin, a residual concentration of 60% was still present after a light irradiance of 3470 kJ/m(2), by means of a 250 W/m(2) light power for 240 min, versus a residual value of just 8% measured at the same time in ethanol solution. Moreover, the isomerization rate in liposomes resulted reduced for isotretinoin and practically irrelevant for tretinoin. The degradation rate was found to be dependent on the drug concentration. The better stability of the tretinoin in liposome complex was supposed to be related to its higher incorporation value due to the linear structure of the molecule.

Niosomes as carriers for tretinoin. II. Influence of vesicular incorporation on tretinoin photostability.

In this work, we compared the chemical stability of tretinoin (TRA) in methanol and in vesicular suspensions exposed both to UV and artificial daylight conditions with the aim of evaluating the potential of niosomes as topical carriers capable of improving the stability of photosensitive drugs. Tretinoin-loaded niosomes were prepared from polyoxyethylene (4) lauryl ether (Brij 30), sorbitan esters (Span 40 and Span 60) and a commercial mixture of octyl/decyl polyglucosides (Triton CG110). Liposomes made from hydrogenated (P90H) and non-hydrogenated (P90) soy phosphatidylcholines were also prepared and studied. In order to evaluate the influence of vesicle structure on the photostability of tretinoin, TRA-loaded vesicles were prepared by the film hydration method, extrusion technique and sonication. After UV irradiation, TRA dissolved in methanol degraded very quickly while the incorporation in vesicles always led to a reduction of the photodegradation process. The photoprotection offered by vesicles varied depending on the vesicle structure and composition. After fluorescent light irradiation for 21 days, not all the studied vesicular formulations improved TRA stability when compared with the free drug in methanol. Tretinoin incorporated in P90 or Span vesicles presented a half-life shorter or very close to that of the free drug. However, the inclusion of TRA in P90H liposomes and Brij 30 or Triton CG110 niosomes retarded the drug photodegradation.

The stability of tretinoin in tretinoin gel microsphere 0.1%.

Topical tretinoin is highly effective and widely used in the treatment of acne vulgaris. In studies to determine the degree of tretinoin photo degradation (isomerization), 2 tretinoin formulations, tretinoin gel microsphere 0.1% and tretinoin gel 0.025%, alone or in combination with erythromycin-benzoyl peroxide topical gel, were exposed to fluorescent light, incandescent light, or darkness for up to 24 hours. Results of the investigations revealed that after 24 hours of exposure to fluorescent light, 98% of the initial tretinoin in the tretinoin gel microsphere 0.1% formulation remained unchanged. When tretinoin gel microsphere 0.1% was combined with erythromycin-benzoyl peroxide topical gel and exposed to fluorescent light, 99% and 87% of the tretinoin was recovered after 4 and 24 hours, respectively, indicating only a limited amount of degradation. In contrast, exposure of tretinoin gel 0.025% to 24 hours of fluorescent light resulted in up to 69% tretinoin degradation and up to 89% degradation when the gel was combined with the erythromycin-benzoyl peroxide topical gel. The data suggest that the tretinoin gel microsphere 0.1% formulation offers marked protection against tretinoin photo degradation, even in the presence of a strong oxidizing agent such as benzoyl peroxide.

Photochemical and oxidative degradation of the solid-state tretinoin tocoferil.

The photostability of tretinoin tocoferil was investigated under irradiation with three kinds of lamps, i.e., a cool white fluorescent lamp, a UV-A fluorescent lamp and a D65 fluorescent lamp. A combination of the cool white fluorescent lamp and the UV-A fluorescent lamp, and the D65 lamp having relative spectral power distribution similar to that of direct daylight, correspond to options 2 and 1 in ICH Guidelines, respectively. The photodegradation apparently followed second-order kinetics under these light sources and the degradation rate constant under exposure by the D65 lamp was larger than that by the cool white fluorescent lamp. The drug was susceptible to degradation by visible and UV light below 480 nm and was degraded most remarkably at around 420 nm, showing a wavelength-dependency. The semi-logarithmic plots of apparent degradation rate constant against the reciprocal of illuminance showed a good linear relationship in the Arrhenius-type fashion, and the photostability under ordinary illumination conditions could be predicted from the data obtained under the accelerated illumination conditions. The rate of oxidative degradation was slightly accelerated with the rise of temperature. Thermodynamic parameter was calculated from the Arrhenius plot. The degradation rate constant rapidly increased in proportion to partial pressure of oxygen below 20 kPa.

Photoisomerization of retinoic acid and its influence on regulation of human keratinocyte growth and differentiation.

Retinoic acid constantly undergoes structural inter-conversions among the geometrical isomers (all-trans-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid and 9-13-di-cis-retinoic acid) by photoisomerization under natural light. Geometric isomers of retinoic acid thus formed showed different effects on human epidermal keratinocyte growth and differentiation. The ability of the isomers to inhibit the synthesis of cornified envelope (terminal event in the keratinocyte differentiation program) changed rapidly when illuminated by white fluorescent light. The 11-cis-retinoic acid had a 3-fold stronger activity to inhibit the growth of keratinocytes than the other geometric isomers. On the other hand, all-trans-retinoic acid, 9-cis-retinoic acid and 9-13-di-cis-retinoic acid exhibited a 3-fold greater ability to inhibit synthesis of involucrin, transglutaminase and the cornified envelopes. The regulation of keratin expression by the geometric isomers of retinoic acids was extremely complex. Level of keratin-1 (K1) mRNA was increased by 11-cis-retinoic acid and 13-cis-retinoic acid, but suppressed by 9,13-di-cis-retinoic acids while all-trans-retinoic acid and 9-cis-retinoic acid had no effect. Level of keratin-10 (K10) mRNA was strongly inhibited by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid as compared to 13-cis-retinoic acid and 9,13-di-cis-retinoic acids. The mRNA level of keratin-14 (K14) was suppressed by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid but not influenced by 13-cis-retinoic acid and 9,13-di-cis-retinoic acid. Natural light induced structural inter-conversions among the geometric isomers of retinoic acids in tissues-especially the skin, might play a crucial role in the regulation of growth and differentiation of keratinocytes.

Investigation on the photostability of a tretinoin lotion and stabilization with additives.

Tretinoin, a drug that is used in topical preparations for the treatment of acne vulgaris, is known to be very susceptible to degradation under daylight. The objective of this work was to investigate the degradation of a tretinoin lotion placed in front of a xenon lamp. Analysis was performed with HPLC. The tretinoin lotion was degraded to about 20% of its initial concentration within 30 min. Incorporation of tretinoin in beta-cyclodextrin or in some surfactants (Brij(R)s) did not have any effect on the photodegradation of tretinoin. Neither could a UV-B sunscreen retard the photodegradation of tretinoin while a UV-A sunscreen had very little effect. Irradiation with selected wavelengths revealed that 420 nm seemed to be the most harmful wavelength for the degradation of tretinoin and not the wavelength of maximum absorption (350 nm) as expected. Then the addition of the yellow colourants chrysoin and fast yellow, absorbing in the region of 420 nm, was tested. These colourants did indeed retard the photo-degradation of tretinoin more or less depending on the concentration of the dye. Finally we only had to select a concentration that was still effective but that did not colour the skin.

Chemical stability of adapalene and tretinoin when combined with benzoyl peroxide in presence and in absence of visible light and ultraviolet radiation.

Adapalene and tretinoin are molecules used in the topical treatment of acne vulgaris. Commercial formulations (adapalene 0.1% gel and tretinoin 0.025% gel) were mixed with equal volumes of commercially available benzoyl peroxide formulation (10% lotion) and subsequently exposed to light over 24 h. With and without exposition to light, adapalene exhibits a remarkable stability whereas tretinoin is very sensitive to light and oxidation. The combination of benzoyl peroxide and light results in more than 50% degradation of tretinoin in about 2 h and 95% in 24 h.

Molecular flexibility of retinoic acid under white fluorescent light.

Among the series of metabolic analogs of the eccentric cleavage pathway of beta-carotene with different side chain lengths, retinoic acid was shown to have exceedingly higher molecular flexibility to undergo photoisomerization into the geometrical isomers under white fluorescent light. When irradiated with white fluorescent lamps (1,200 lx), the velocity of photoisomerization of all-trans-retinoic acid (8.4 x 10(-7) mol/L.min) was exceedingly higher than those of the other analogs: ionylideneacetic acid (4 x 10(-8) mol/L.min), ionylidenecrotonic acid (3.0 x 10(-7) mol/L.min), all-trans-beta-apo-14'-carotenoic acid (1.7 x 10(-7) mol/L.min), all-trans-beta-apo-12'-carotenoic acid (1.3 x 10(-7) mol/L.min), and all-trans-beta-apo-8'-carotenoic acid (0.1 x 10(-7) mol/L.min). beta-Carotene did not undergo photoisomerization under the experimental conditions. The molecular flexibility of retinoic acid is assumed to be an important basis of the mechanism of action of retinoic acid.

A fast photoisomerization method for the preparation of tritium-labeled 9-cis-retinoic acid of high specific activity.

A method is described by which tritium-labeled all-trans retinoic acid of high specific activity (up to approximately 51 Ci/mmol corresponding to 85% of theoretical) is converted photolytically within a fraction of a second to a mixture of retinoic acid stereoisomers. One of these isomers, 9-cis-retinoic acid, was obtained in high radiochemical purity by reverse-phase HPLC of the stereoisomer mixture. This fast photolysis was obtained by using a high-pressure 100-W mercury lamp operated at 86 +/- 2 W. A copper sulfate solution was used as a light filter to eliminate short-wave ultraviolet radiation as well as much of the infrared radiation. The geometry of the experimental set-up allowed a maximal amount of the light output of the lamp to reach the retinoic acid solution. Reverse-phase HPLC of the photolytically generated retinoic acid stereoisomer mixture provided pure 9-cis-retinoic acid in 4.5% yield after irradiation for 0.6 s. A steady-state equilibrium of retinoic acid stereoisomers was reached when the irradiation time was extended to a total of 4-6 s (10-11% yield of 9-cis retinoic acid).

Normal phase LC-MS determination of retinoic acid degradation products.

The degradation products formed when 13-cis retinoic acid (13-cis RA) and all-trans RA were exposed to fluorescent light and air were investigated. These retinoids are known to undergo Z-E isomerization (due to the existence of four unsaturated double bonds) and oxidation when exposed to light and air. Analysis by LC was carried out on a 25 cm x 4.6 mm Zorbax Rx-SIL (5 microns) with a mobile phase (1.4 ml min-1) of heptane-THF-acetic acid (96.5:3.5:0.015) and an in-line UV (365 nm) detector. The LC eluate was coupled through a Vestec universal interface to a Finnigan 4023 mass spectrometer. EI-mass spectra were obtained at 77 eV from m/z 200 to 350 with multiplier voltage of 1200 V. Solid samples of 13-cis RA and all-trans RA exposed to light and air and also solutions of these retinoids in the mobile phase exposed to the same conditions were used for the analysis. Tentative identities of the degradation products from the mass spectra suggest the isomerization of the retinoids (Z-E isomerism) and the formation of the 5,6-epoxides of these isomers. Identities of the 5,6-epoxides were confirmed with chromatographic and mass spectral data from synthetic samples of the epoxides. Isomerization occurred more readily in solution than in the solid form and the 13-cis RA isomer oxidized more readily than the all-trans isomer.

High-performance liquid chromatographic separation and spectral identification of the photoreaction products of methyl 5,8-epoxyretinoate.

Photoreaction of methyl 5,8-epoxyretinoate was investigated. Irradiation of methyl 5,8-epoxyretinoate in acetonitrile with a light from a high-pressure mercury lamp or a daylight fluorescent lamp afforded three new products, which were purified by high-performance liquid chromatography. They were characterized on the basis of spectroscopic data as 11-(Z), 13-(Z), and 11,13-di(Z) isomers, respectively.