Can Mycosporine‐Like Amino Acids Act as Multifunctional Compounds in Gymnodinium catenatum (Dinophyceae)?

MAAs originating from Gymnodinium catenatum were subjected to H₂O₂ oxidation, light and heat. Shinorine and porphyra‐334 were the more resistant to all treatments, mycosporine‐glycine (MYGL) was the least resistant to oxidation and heat, whereas palythene and M‐370 were the least resistant to light. MYGL and M‐311 were similarly resistant to photodegradation and oxidation in the dark and low temperature, but M‐311 was more resistant to oxidation under light or heat. The ratio M‐370/M‐365 changed from 29:1 to 6:1 ratio after 240 h of exposure to fluorescent light, indicating that M‐365 could represent the M‐370 cis‐isomer. The role of MAAs as antioxidants and/or osmolytes was evaluated by studying effects of abrupt salinity reduction. Both increases or decreases in concentrations were observed and were dependent on the MAA initial concentration and its chemical structure. The relative increase in MAAs with a known antioxidant capacity (MYGL, palythene) followed an exponential decay trend related to initial concentration. The relative decrease in highly polar MAAs (shinorine, porphyra‐334, M‐332) with a suspected osmolyte role followed a rise to a maximum with the increase in initial concentration. Whether or not MAAs play a significant role in osmoregulation, their loss can occur upon hypoosmotic shock.     

Effects of Salinity and Ultraviolet Radiation on the Bioaccumulation of Mycosporine‐like Amino Acids in Artemia from Lake Urmia (Iran)

We investigated the effects of salinity and artificial UV radiation on the accumulation of mycosporine‐like amino acids (MAAs) in sexual and parthenogenetic Artemia from Lake Urmia. The nauplii hatched from the cysts were cultured until adulthood under two salinities (150 and 250 g L^?1) and two light treatments (PAR and PAR+UVR) in the laboratory. Finally, the Artemia were analyzed for their concentration of MAAs. In most of the cases, the higher salinity level applied was found to increase the MAA concentrations in both Artemia populations significantly. The acquisition efficiency of MAAs in both Artemia populations increased under exposure to UVR‐supplemented photosynthetically active radiation (PAR) compared to those raised under PAR, except for Porphyra‐334. It was observed that combination of UV radiation and elevated salinity significantly increased the bioaccumulation of MAAs. Thus, the presence of these compounds in these populations of Artemia may increase their adaptability for living in high‐UV and high‐salinity conditions prevailing in Lake Urmia. Higher concentrations of MAAs in the parthenogenetic population of Artemia could be probably attributed to its mono sex nature and higher adaptation capacities to extreme environmental conditions.     

Biotransformation of mycosporine like amino acids (MAAs) in the toxic dinoflagellate Alexandrium tamarense

Changes in mycosporine-like amino acids (MAAs) induced by the increase of photosynthetically active radiation (PAR) were studied in the toxic dinoflagellate Alexandrium tamarense. Cultures of A. tamarense were maintained at exponential growth under low (25 micromol quanta m(-2)s(-1)) PAR irradiance. The cultures were nutrient enriched and one day later exposed to higher irradiance (150 micromol quanta m(-2)s(-1)). The content of MAAs was determined by means of high performance liquid chromatography (HPLC). Eleven MAAs, including some partially characterized compounds, were identified. The MAAs synthesis induction can be described as a two-stage process. The first one involves the net synthesis of the MAAs bi-substituted by amino acids. In the second stage these compounds were transformed into other secondary MAAs. The two most prominent changes were observed in the concentration of porphyra-334 and palythene. The cellular concentration of porphyra-334 increased during the first 2h of exposure to higher irradiance and then decreased rapidly. In contrast, the cellular concentration of palythene showed a continuous accumulation since the beginning of the exposure. In A. tamarense the main route of MAAs transformation has porphyra-334 as a precursor of a sequential conversion resulting in the accumulation of palythene.     

Mycosporine-like Amino Acids Provide Protection Against Ultraviolet Radiation in Eggs of the Green Sea Urchin Strongylocentrotus droebachiensis

A photoprotective role of ultraviolet radiation-absorbing mycosporine-like amino acids (MAAs) in eggs of the green sea urchin Strongylocentrotus droebachiensis was demonstrated by comparing UV-induced delays in the first division of embryos having either high or low concentrations of MAAs. Embryos from adult urchins fed Laminaria saccharina (no MAAs) had low concentrations of MAAs and experienced a significantly longer UV-induced delay in cleavage (25.1%) than MAA-rich embryos from adults fed Mastocarpus stellatus (12.8% delay) or a combination diet of both macroalgae (12.3% delay). Collectively, these embryos displayed a significant inverse logarithmic relationship between MAA concentration and percentage cleavage delay, so that the greater the MAA concentration in the eggs, the less they were affected by UV radiation. This is the first study to examine such MAA manipulation of cellular MAA concentrations with no prior UV exposure of the experimental subjects. Concentrations of MAAs were also measured in unfertilized eggs, blastulae, gastrulae and early pluteus larvae, providing the first documentation of changes in MAAs during embryological and larval development. The concentration of shinorine (the principal MAA in the eggs) did not change during short-term UV exposure in vivo or long-term exposure in vitro; such photostability is a useful attribute of a natural sunscreen.     

Effects of short-term irradiation on photoinhibition and accumulation of mycosporine-like amino acids in sun and shade species of the red algal genus Porphyra

The effect of irradiance (40 and 840 micromol photons m(-2) s(-1)) of short-term (48 h) irradiation on photosynthetic activity (estimated as oxygen evolution and as chlorophyll fluorescence), specific absorption and fluorescence excitation spectra, photosynthetic pigment accumulation (chlorophyll a and biliproteins) and UV-absorbing compounds (mycosporine-like amino acids, MAAs) was investigated in sun and shade species of the red algal genus Porphyra collected in Trondheimsfjord (Norway). In the sun type, high irradiance exposure (840 micromol photons m(-2) s(-1)) did not alter the Chl a concentration, however, exposure to a lower irradiance (40 micromol photons m(-2) s(-1)) for 48 h significantly increased the chlorophyll concentration. The content of MAAs was significantly higher in the suntype than in the shade type algae. Porphyra-334 is the main MAA in this species followed by shinorine. The total content of MAAs significantly (P<0.05) increased in the sun type after 48 h exposure to both high and low irradiances. However, in the shade type, porphyra-334 significantly decreased (P<0.05) after both high and low irradiance exposure. Photosynthetic activity (as oxygen evolution) and the optimal quantum yield (F(v)/F(m)), as an indicator of photoinhibition, decreased under low and high irradiance in the shade type algae and no full recovery was observed when the algae were transferred to very low irradiation.The sun type algae presented a higher capacity of acclimation to increased irradiance than the shade type algae. This high acclimation of sun type algae to short term high irradiance exposure (48 h) is explained by the higher thermal dissipation. This was estimated as the ratio of nonphotochemical quenching related to the light dose (q(N):dose) and by the accumulation of MAAs.     

Electrospray ionization tandem mass spectrometric and electron impact mass spectrometric characterization of mycosporine-like amino acids

Positive-ion mass spectral fragmentations of seven mycosporine-like amino acids (MAAs) are reported and discussed. The MAAs studied are small compounds composed of a cycloheximine ring substituted with amino acid or amino alcohol units. Techniques used include electron impact (EI) and electrospray ionization (ESI) with tandem mass spectrometry (MS/MS). ESI-MS/MS showed unusual small radical losses, generally resulting from the loss of a methyl group with the exception of shinorine and porphyra for which the initial losses were 30 and 44 Da, respectively. As expected from structural similarities, porphyra, shinorine and palythinol displayed similar fragmentation patterns, while palythenic acid and palythene fragmented in a similar manner. Overall, the ESI-MS/MS fragmentations at m/z <200 exhibited a distinctive pattern for all seven MAAs with characteristic ions at m/z 137, 168, 186, and 197 or 199. Several ions were observed for each of the MAAs analyzed, and together provide a useful and potentially diagnostic pattern for identification of MAAs and as an aid in structure elucidation of novel MAAs. For GC/EI-MS analysis, trimethylsilyl (TMS) derivatives were made. The EI-MS fragmentation patterns of TMS-MAAs showed many features typical of TMS-derivatized alpha-amines. The precursor TMS-MAA ion was not detected, but a [M-90](+ radical) ion was the highest-mass intense peak observed for palythine, palythinol and shinorine, while palythene gave a [M-116](+ radical) ion. Besides determining the number of acidic hydrogens, EI-MS of TMS-derivatized MAAs will aid in structure elucidation of novel MAAs.     

Mycosporine-like Amino Acids in Freshwater Copepods: Potential Sources and Some Factors That Affect Their Bioaccumulation

Mycosporine-like amino acids (MAAs) are ubiquitous photoprotective compounds in aquatic environments. MAAs are synthesized by a wide variety of organisms (i.e. bacteria, fungi and algae) and their production is photoinducible by ultraviolet radiation (UVR) (280–400 nm) and/or photosynthetically active radiation (400–750 nm). Most animals however, are unable to synthesize MAAs and must acquire these compounds through their diet or from symbiotic organisms. In this paper, we investigate the possible sources of MAAs and factors (temperature and initial MAA concentration) that may affect their bioaccumulation in freshwater copepods. We found that MAA accumulation may occur even if the copepods are cultured on a MAA-free diet. In addition, we found that the bacteriostatic antibiotic, chloramphenicol, inhibits the bioaccumulation of MAAs. These two pieces of evidence suggest that the source of MAAs in these copepods may be prokaryotic organisms in close association with the animals. The two factors investigated in this study, temperature and initial MAA concentrations, were found to affect the rates at which MAAs are accumulated. Temperature had positive effects on both uptake and elimination rates. On the other hand, the rate of uptake decreased at the highest assayed initial MAA concentration, probably because the concentration of MAAs was already close to saturation.     

Cellular Changes Associated with the Acclimation of the Intertidal Sea Anemone Actinia tenebrosa to Ultraviolet Radiation

To assess the relative importance of long‐ and short‐term cellular defense mechanisms in seasonally UV‐R‐acclimated Actinia tenebrosa (Anthozoa, Actiniidae), individuals were exposed to summer doses of PAR, UV‐A, UV‐B and enhanced UV‐B (20%) for a period of 4 days. Mycosporine‐like amino acids (MAAs) and cyclobutane pyrimidine dimer (CPD) concentrations were quantified, while oxidative damage to lipids and proteins, and the activities or levels of the antioxidant enzymes SOD, CAT, GR, GPOX and total glutathione were determined. Our results show that summer UV‐R‐acclimated individuals had a higher UV‐R tolerance, with no significant increases in CPDs levels, than winter‐acclimated sea anemones possibly due to higher MAA concentrations. Summer‐acclimated individuals showed increased lipid and protein oxidation and GPOX activity only when they were exposed to UV‐B at 20% above ambient UV‐R levels. In contrast, winter‐acclimated sea anemones showed elevated levels of oxidative damage, GPOX and SOD activities after exposure to UV‐A or UV‐B at ambient and elevated levels. Thus, this study indicates that long‐term UV‐R acclimation mechanisms such as the accumulation of MAAs could be more important than short‐term increases in antioxidant defenses with respect to reducing indirect UV‐R damage in intertidal sea anemones.     

Daily variation in cellular content of UV-absorbing compounds mycosporine-like amino acids in the marine dinoflagellate Scrippsiella sweeneyae

Sudden exposure experiments to high PAR (photosynthetically available radiation) or high PAR+UVR (ultraviolet radiation) were conducted for the marine dinoflagellate Scrippsiella sweeneyae acclimated to either low PAR or high PAR to determine the induction of cellular mycosporine-like amino acid (MAA) in relation to photosynthesis status. When the exposure to high PAR (30.8 Wm(-2)) was provided at different time in the light period for S. sweeneyae acclimated to low PAR (7.7 Wm(-2)) which suppressed photosynthesis, S. sweeneyae could enhance the induction of MAA but it only occurred in the first half of the light period. When UVR exposure was provided for the culture acclimated to high PAR which enhanced photosynthesis, cellular MAA content did not increase during the entire light period, but displayed daily variation similar to the control for two and half days. Daily variation of cellular MAA content did not synchronized with that of cell volume and cellular chlorophyll a content. The individual MAAs also revealed similar daily variations with different phase, which increased for a few hours in the beginning of the light period, except for cellular palythine content. Thus the total cellular MAA content revealed daily variation with changing the relative composition within a few hours. As one of the biological protective strategies against harmful UVR in sunlight, the daily vertical migration in the bloom forming dinoflagellates might be accompanied by the daily variation of cellular MAA content for a photosynthesis at daytime.     

Mycosporine-like amino acids in the marine dinoflagellate Gyrodinium dorsum: induction by ultraviolet irradiation

Cultures of the marine dinoflagellate Gyrodinium dorsum have been exposed to polychromatic radiation (photosynthetically active radiation and UV) from a solar simulator for up to 72 h. Different irradiance spectra in the ultraviolet are produced by inserting cut-off filters between lamp and samples. The mycosporine-like amino acid (MAA) content and composition are investigated by spectroscopic and chromatographic analysis. The study reveals that G. dorsum contains a complex mixture of several aminocyclohexenimine-MAAs and one aminocyclohexenone-MAA. UV irradiation around 320 nm induces an increase in the concentration of all MAAs in the samples. In contrast, exposure to short-wavelength UV-B radiation results in decreased overall MAA production. Furthermore, there is a spectral shift in the absorption of the MAA mixture towards shorter wavelengths, indicating that short-wavelength UV-B induces an altered MAA composition. The amount of MAAs is normalized to the chlorophyll a concentration.     

Novel glycosylated mycosporine-like amino acids with radical scavenging activity from the cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are UV absorbing pigments, and structurally distinct MAAs have been identified in taxonomically diverse organisms. Two novel MAAs were purified from the cyanobacterium Nostoc commune, and their chemical structures were characterized. An MAA with an absorption maximum at 335 nm was identified as a pentose-bound porphyra-334 derivative with a molecular mass of 478 Da. Another identified MAA had double absorption maxima at 312 and 340 nm and a molecular mass of 1,050 Da. Its unique structure consisted of two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen and two pentose and hexose sugars. These MAAs had radical scavenging activity in vitro; the 1050-Da MAA contributed approximately 27% of the total radical scavenging activities in a water extract of N. commune. These results suggest that these glycosylated MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune.     

Photodegradation and photosensitization of mycosporine-like amino acids

The photodegradation and photosensitization of several mycosporine-like amino acids (MAAs) were investigated. The photodegradation of the MAA, palythine, was tested with three photosensitizers: riboflavin, rose bengal and natural seawater. For comparison of degradation rates, the riboflavin-mediated photosensitization of six other MAAs was also examined. When riboflavin was used as a photosensitizer in distilled water, MAAs were undetectable after 1.5h. Palythine showed little photodegradation when rose bengal was added as the photosensitizer (k=0.12x10(-3)m(2)kJ(-1)). Palythine dissolved in natural seawater containing high nitrate concentrations also showed slow photodegradation rate constants (k=0.26x10(-3)m(2)kJ(-1)) over a 24-h period of constant irradiation. Similar experiments in deep seawater with porphyra-334 and shinorine resulted in 75% of the initial MAA remaining after 4h of irradiation and rates of 0.018 and 0.026x10(-3) m(2) kJ(-1), respectively. Experiments conducted in deep seawater with riboflavin additions resulted in photodegradation rate constants between 0.77x10(-3) and 1.19x10(-3)m(2)kJ(-1) for shinorine and porphyra-334, respectively. Photoproduct formation appeared to be minimal with the presence of a dehydration product of the cycloheximine ring structure indicated as well as the presence of amino acids. Evidence continues to build for the role of MAAs as potent and stable UV absorbers. This study further highlights the photostability of several MAAs in both distilled and seawater in the presence of photosensitizers.     

In vitro cis-trans photoisomerization of palythene and usujirene. Implications on the in vivo transformation of mycosporine-like amino acids

The in vitro photoinduced reactions of the mycosporine-like amino acids (MAA) usujirene and palythene were studied by monochromatic stationary irradiation at 366 nm. High-performance liquid chromatography analysis of the irradiated aqueous solution of usujirene indicated a low photoreactivity on the basis of the observed photodecomposition quantum yield of phi(-U) = (2.86 +/- 0.80) x 10(-5), which can be partially accounted for by the cis-trans photoisomerization of usujirene to palythene (phi(U-->P) = [1.71 +/- 0.13] x 10(-5)). However, palythene in aqueous solution showed a higher photostability than did usujirene under equivalent conditions, establishing a photostationary mixture of cis-trans isomers with a relative composition of palythene-usujirene (11:1). These results may explain the preferential in vivo accumulation of palythene relative to that of usujirene observed in several dinoflagellate species.     

Photoprotection and long-term acclimation to UV radiation in the marine diatom Thalassiosira weissflogii.

Unialgal cultures of the marine diatom Thalassiosira weissflogii (Grunow) were exposed for 40 days to artificial UV-B radiation in the presence of PAR and UV-A to examine long-term acclimation to UV, PAR and UV-A were supplied 14 h daily, while UV-B (two levels: 0.16 and 0.30 W m(-2) unweighted) was supplied for 4 h/day. Growth rates and photochemical capacity (CFC ratio) both decreased over the first 10-15 days, then recovered. No obvious differences were noted between the responses to the two UV-B treatments. The concentration of the major pigments (chlorophyll a and c(1+2), fucoxanthin and beta,beta-carotene) changed very little with time, except for diatoxanthin. which increased over the first 16 days, decreased over the next 13 days, then increased again from day 29 to the end of the experiment. The concentration of total mycosporine-like amino acids (MAAs) was initially undetectable, then increased from day 16 in the high UV-B treatment and after day 22 in the low UV-B treatment, reaching a maximum on day 29 for both treatments and decreasing afterwards. The synthesis of MAAs proceeded only once photochemical capacity had recovered from the initial UV stress and this recovery likely involved the xanthophyll cycle (diatoxanthin increase). The concentration of MAAs decreased when the cells showed signs of photoinhibition (decrease in CFC ratio). It also showed an inverse trend with diatoxanthin. UV-B alone had little regulatory effect over these responses, except possibly for an earlier synthesis of MAAs under HUV-B conditions. This suggests that the observed changes were due to UV-A rather than to UV-B exposure. The overall response of this coastal diatom to prolonged UV exposure indicates that T. weissflogii is a relatively UV-tolerant species and that its long-term response to UV exposure involves an activation of the xanthophyll cycle followed by the synthesis of MAAs, which may proceed only when photoinhibition is relieved.     

Induction of mycosporine-like amino acids (MAAs) in cyanobacteria by solar ultraviolet-B radiation

Three filamentous and heterocystous N₂-fixing cyanobacteria, Anabaena sp., Nostoc commune and Scytonema sp. were tested for the presence of ultraviolet-absorbing mycosporine-like amino acids (MAAs) and their induction by solar ultraviolet-B (UV-B) radiation. High performance liquid chromatographic (HPLC) studies revealed the presence of only one type of MAAs in all three cyanobacteria, that was identified as shinorine, a bisubstituted MAA containing both glycine and serine groups having an absorption maximum at 334 nm and a retention time of around 2.8 min. There was a circadian induction in the synthesis of MAAs when the cultures were exposed to mid-latitude solar radiation (Playa Unión, Rawson, Chubut, Patagonia, Argentina) for 3 days, 4–6th February, 2000. Solar radiation was measured by an ELDONET (European Light Dosimeter Network) filter radiometer permanently installed on the roof of the Estación de Fotobiología Playa Unión (43°18′ S; 65°03′ W). The maximum irradiances were around 450–500, 45–50 and 1.0–1.2 W m⁻² for PAR (photosynthetic active radiation), UV-A (ultraviolet-A) and UV-B (ultraviolet-B), respectively. PAR and UV-A had no significant impact on MAA induction while UV-B induced the synthesis of shinorine in all three cyanobacteria. Shinorine was found to be induced mostly during the light period. During the dark period the concentration stayed almost constant. In addition to shinorine, another unidentified, water-soluble, brownish compound with an absorption maximum at 315 nm was found to be induced by UV-B only in Scytonema sp. and released into the medium. This substance was neither found in Anabaena sp. nor in Nostoc commune. Judging from the results, the studied cyanobacteria may protect themselves from deleterious short wavelength radiation by their ability to synthesize photoprotective compounds in response to UV-B radiation.     

Synthesis and aggregation behaviour of amphiphilic block copolymers with random middle block

The effect of microstructure on the aggregation behaviour of symmetrical di- and triblock copolymers P(BMA)-b-P(MAA) and P(BMA)-b-P(BMA-co-MAA)-b-P(MAA) with a molecular weight of 40,000 g/mol was studied. The critical micelle concentration, hydrodynamic radius and morphology of the micelles were determined by fluorescence spectroscopy, dynamic light scattering and scanning force microscopy (SFM). Whereas no effect of the microstructure on the critical micelle concentration could be detected, the hydrodynamic radius decreased from di- to triblock copolymer from 53 to 36 nm. The decrease of about 32% corresponds to the length of the random middle block within the triblock copolymer so that the reduction in hydrodynamic radius was caused by a complete orientation of the random middle block at the core corona interface. Finally, the SFM investigation showed that dehydration of micelles on a substrate is accompanied by formation of a physisorbed monolayer with a thickness of 2 nm on which the micelles are deposited.     

Using flow field-flow fractionation (Fl-FFF) for observation of salinity effect on the size distribution of humic acid aggregates

Flow field-flow fractionation (Fl-FFF) was used to investigate the effect of salinity on the size distribution of humic acid (HA) aggregates in estuarine water. In water with high salinity as estuarine water, size distributions were slightly broadened with increasing contact time between HA and estuarine water. At the longest contact times (89 days) and highest salinity value (28 psu, g kg^?1), the peak maxima were observed at 1.7 and 8.6 nm when detected at 254 nm, and at 1.9 and 9.1 nm when detected at 400 nm. In addition, Fl-FFF with an inductively coupled plasma mass spectrometry was applied to examine the effect of salinity on the size distribution of Cd, Ce, Cu, Mn and Pb-binding HA aggregates in estuarine water with different salinity values. At 1 day contact time, the peak maxima of Cd, Ce, Cu, Mn and Pb-binding HA aggregates in water with increased salinity values were increased and gave the larger breadth of size distribution. The larger size fraction of HA aggregates showed more affinity for Pb, Cd, Ce and Mn than Cu whereas the smaller size fraction of humic aggregates showed preferential binding towards Cu.     

Wavelength dependence of mycosporine-like amino acid synthesis in Gyrodinium dorsum

The synthesis or accumulation of mycosporine-like amino acids (MAAs) is an important UV tolerance mechanism in aquatic organisms. To investigate the wavelength dependence of MAA synthesis in the marine dinoflagellate Gyrodinium dorsum, the organism was exposed to polychromatic radiation (PAR and UV) from a solar simulator for up to 72 h. Different irradiance spectra were produced by inserting various cut-off filters between lamp and samples. A polychromatic action spectrum for the synthesis of MAA synthesis was constructed. PAR and long wavelength UV-A radiation showed almost no effect while the most effective wavelength range was around 310 nm. Shorter wavelengths where less effective in the induction of MAA synthesis. Wavelengths below 300 nm damaged the organisms severely as indicated by a decrease in chlorophyll a absorption.     

Photocatalytic patterning of monolayers for the site-selective deposition of quantum dots onto TiO2 surfaces

A novel photochemical mechanism is reported for the site-selective deposition of quantum dots onto nanocrystalline TiO2 films. The patterning mechanism involves the combination of surfactant-mediated self-assembly and monolayer photolithography. In the self-assembly process, CdS and CdSe quantum dots were attached to TiO2 surfaces through bifunctional mercaptoalkanoic acid (MAA) linkers. MAAs were adsorbed to the TiO2 surface as the deprotonated carboxylates, primarily through monodentate coordination to Ti4+ sites. CdSe quantum dots were bound to the terminal thiol groups of surface-adsorbed MAAs, with a surface adduct formation constant, Kad, of (2.1 +/- 0.7) x 104 M-1. The color and optical density of the quantum dot-functionalized TiO2 films were tunable. Monolayer photopatterning involved the TiO2-catalyzed oxidative degradation of surface-adsorbed mercaptohexadecanoic acid (MHDA). A mechanism is proposed wherein MHDA degradation occurs through both oxidative decarboxylation and the formation of interchain disulfides. These MHDA photodegradation processes regulate the extent to which CdSe quantum dots adsorb onto the TiO2 surface. Illumination through a photomask yielded optically patterned, quantum dot-functionalized TiO2 films that were characterized by scanning electron microscopy and energy-dispersive X-ray analysis.     

Identification and characterization of forced degradation products and stability‐indicating assay for notoginsenosidefc by using UHPLC‐Q‐TOF‐MS and UHPLC‐MS/MS: Insights into stability profile and degradation pathways

Notoginsenoside Fc, a protopanaxadiol‐type saponin, shows multi‐pharmacological activities. Chemical stability evaluation plays a crucial role in drug development. In this study, the forced degradation behavior of Notoginsenoside Fc was investigated under hydrolytic and oxidative conditions. A specific ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the separation, identification, and characterization of the degradation products of Notoginsenoside Fc. Fifty potential degradation products were formed via deglycosylation, dehydration, hydration, isomerization, side‐chain cleaving, oxidation, and superoxidation. Notoginsenoside Fc was subjected to different pH solutions, temperatures, and time periods to assess its stability. A sensitive ultra high performance liquid chromatography‐tandem mass spectrometry was developed for the quantification of Notoginsenoside Fc, notoginsenoside ST‐4, notoginsenoside Ft1, and relative quantification of notoginsenoside Ft2, 20(R)‐notoginsenoside Ft2, notoginsenoside SFt3, and notoginsenoside SFt4. The assay was linear over the concentration range (R² > 0.997) with the lowest limit of quantification of 0.02 μg/mL for Notoginsenoside Fc, Notoginsenoside ST‐4, and Notoginsenoside Ft1. The intra‐day precision, inter‐day precision, and accuracy of the three analytes were within accepted levels. The degradation kinetics of Notoginsenoside Fc in pH 1 and 3 solutions fits to first‐ and second‐order kinetics, respectively. The degradation of Notoginsenoside Fc is pH‐, temperature‐, and time‐dependent.