The Profile Of Carotenoids By LC-MS And Its Application In Distribution Metabolism Of Mytilus Coruscus

Carotenoids are widely found in aquatic organisms,with the functions of organization coloring,improving environmental adaptation,participating in free radical clearance,enhancing reproductive function and improving body immunity.Aquatic organisms can absorb and transform exogenous carotenoids into their own tissue-specific carotenoids.Influenced by the regulation of the own genetic mechanism and the differences in exogenous bait,it leads to different carotenoid deposition and metabolic transformation in different aquatic animals and tissue sites.In order to explore the metabolism pathway of carotenoid in aquatic products,we took Mytilus coruscus as the research object,and analyzed the effects of different exogenous diets on the carotenoid composition with different growth stage and tissue from four aspects of comprehensive extraction,efficient separation,substance identification and content distribution.The biosynthetic pathway of carotenoid in Mytilus coruscus was preliminarily speculated,which provided technical support for selective cultivation of high-content Mytilus coruscus.The results are as follows:1.A study on sequential extraction and purification of carotenoids from aquatic animalsA system of sequential extraction and purification coupled with UHPLC-Q-Orbitrap-HRMS detection applied to the analysis of three common carotenoids with very different physicochemical properties in aquatic animals was established.In the first-stage of extraction,the free carotenoids were extracted with methanol（0.1%BHT）and purified effectively by HLB,in the second-stage of extraction,the esterified carotenoids were mainly extracted with methanol-dichloromethane（0.1%BHT）and purified effectively by Alumina-N.The quantification limit was 0.05 mg/kg,the recoveries more than 86.9%.In total,nearly 50 carotenoid species were identified in common aquatic animals by combining the first-stage and second-stage solutions.Besides,astacin 22:6/16:0,oxidized-astaxanthin 22:6/16:0 and adonixanthin 22:6/20:5 were firstly identified in the Penaeus monodon.2.An improved method for the separation of carotenoids and carotenoid isomers by UHPLC-Q-Orbitrap-HRMSIn the experiment,the different chromatographic columns with different specifications and flow phase were compared.An improved method for the separation of carotenoids and carotenoid isomers by LC-MS was established.We implemented the use of a common Syncronis C18 column with acetonitrile-water（9:1,v:v,containing 10 m M ammonium acetate）+acetonitrile-isopropanol（7:3,V:V）as the flow phase,to successfully separate a mixture of 31 carotenoids standards with different structural properties,including common oxygen-free carotenoids,oxygenated carotenoids,extremely hydrophobic esterified carotenoids.In addition,the method differentiated between three groups of isomeric carotenoids（lycopene/δ-carotene/γ-carotene/ε-carotene/α-carotene/β-carotene,α-cryptoxanthin/β-cryptoxanthin,and zeaxanthin/lutein）by optimizing the gradient profile.3.Profiling of fragments for carotenoid esters in penaeus monodon by UHPLC-Q-Orbitrap-HRMSThe profiling of carotenoid esters,including acyl chain composition and carotenoid skeleton species,was elucidated using ultra-performance liquid chromatography coupled with quadrupole electrostatic field orbital trap high-resolution mass spectrometry（UHPLC-Q-Orbitrap-HRMS）.The mass spectrometry database of carotenoids in common aquatic products were constructed using Exact Finder^TMsoftware.Results showed that carotenoid esters have neutral loss in the form of fatty acids or ketoacids,forming a series of fragmentation fragments,including[MH-RCH₂COOH]⁺,[MH-RCHCO]⁺,[MH-H₂O-RCH₂COOH]⁺and[MH-H₂O-RCHCO]⁺,et al.,which can be used to identify the composition of fatty acyl chain.Notably,the carotenoid skeleton has been identified unequivocally by diagnostic ion*i,*j/*j’and*g/*g’,generated by carbon-carbon bond cleavage betweenβ-ionone ketones and conjugated polyene moieties.The structurally relevant ions*l and*m,can be considered as the markers of the astaxanthin monoester.4.The distribution and metabolism of carotenoids in Mytilus coruscusAccording to the high-throughput screening method established above,we took Mytilus coruscus as the research object.The carotenoid distribution and the expression of associated genes（ABCA1,βCMOOX,βCDIOX and SR-BI）at different larval stage（trochophore,D-shaped larvae,shell stage,eye point phase and juvenile）were analyzed.The results found that there are five carotenoids were identified,including the new synthetic carotenoid mytiloxanthin.Meanwhile,βCMOOX genes associated with carotenoid anabolism,was continuously accumulated with growth,and increased the most rapidly from D-shaped larva to shell stage,with the expression increased 38.2～43.3 times,which suggest thatβCDIOX may be involved in the synthesis of mytiloxanthin during the shell stage.Besides,we carried out feeding experiments with different unialgal cultures（Chaetoceros mulleri and Dicrateria inornata）during the growth stages（proliferation phase,growing phase,mature phase,pre-spawn phase,post-spawn phase,and spent phase）and examined the carotenoid profiles between the dietary microalgae and the corresponding tissues（hepatopancreas,gonad,mantle,and muscle）in order to study the resorption and metabolic transformations of dietary carotenoids in M.coruscus.The results showed that carotenoids mainly occurred in the pre-spawning phase of M.coruscus,the contents of most carotenoids showed the same trends as in dietary microalgae.An association analysis of the total content of carotenoids and relevant gene（ABCA1,βCMOOX,βCDIOX and SR-BI）expression demonstrated that the main tissue for carotenoid synthesis in M.coruscus was the hepatopancreas.The synthetic reactions of the allenic to acetylenic carotenoids pathway and the 4-keto oxidative pathway in M.coruscus coexisted.3)Quantitative analysis revealed that the carotenoids mytiloxanthin,alloxanthin,and pectenolone containing acetylene groups were selectively assimilated in gonad（69.3–74.1%）,mantle（83.2–102%）,and muscle（86.9–100%）,forming a large percentage of the carotenoids present in those tissues.These results provide new insights into the biosynthetic and metabolic transformation pathways of carotenoids in M.coruscus.