Rapid Detection Of Polyamines In Insect By Ultra-performance Liquid Chromatography Tandem Mass Spectrometry

Polyamines(PAs)are aliphatic cations that contain two or more amino groups.They are known to be essential for the growth and development in prokaryotes and eukaryotes.PAs play important roles in the growth of the insect,such as embryonic development and vitellogenin synthesis,growth and metamorphosis,hormone metabolism,and behavior.Although PAs are essential for a variety of physiological and developmental processes in insect,few analytical methods were reported for the detection of PAs in insect.In this study,A fast,simple,and sensitive method for the simultaneous determination of 7 polyamines in Nephotettix cincticeps(N.cincticeps)was developed based on ultra-performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-3Q-MS)together with liquid phase extraction.Then,the proposed method was employed to analyze the change of PAs level in insect under different experiment condition.The results as follows:(1)Polyamines in insect samples were separated and detected by UPLC-3Q-MS,which equipped with a hydrophilic interaction liquid chromatography column without any derivatization procedure.Under the optimized conditions,seven PAs were detected with sharp symmetrical peak shape within 5 min,and the total time for individual sample preparation,separation and detection was about 15 min.The developed method has an instrument LOD of 0.11-0.29 nM,an instrument LOQ of 0.36-0.96 nM,an intra-day RSD of 1.66-4.19%and an inter-day RSD of 1.70-4.72%.The samples were extracted with HClO4 solution,and the recovery rate of spiked samples was around 86-115%with the RSD in the range of 2-6%.(2)The effect of difluormethylornithine(DFMO)on the PAs level in Nephotettix cincticeps.DFMO is an enzyme-activated irreversible inhibitor of ODC,which is the first enzyme in the leading pathway to the biosynthesis of the Put,Spd and Spm in cells.The results showed the level of putrescine,spmrmidine and spmrmine decreased significantly with the increasing of DFMO concentration.On the other hand,the level of other PAs did not show the significant differences.These results demonstrated the developed mothed is capable to correctly determine a change of PAs level in insect.(3)The effect of instar and species of insect on the PAs level.The developed method was applied to detect the PAs concentration of N.cincticeps and Acyrthosiphon Pisum.All the seven PAs with concentration from 1.27 ng/mg(agmatine sulphate)to 314.1 ng/mg(putrescine)were found in N.cincticeps,the most PAs level in N.cincticeps were decreased with the increase of instar,except the level of 1,3-Diaminopropane was increased with the increase of instar,and the level of putrescine reach a maximum in the fourth instar.In addition,the contents of cadaverine,1,3-Diaminopropane and 3,3'-Diaminodipropylamine in female N.cincticeps were significantly higher than those in male N.cincticeps.And only three PAs(putrescine,spmrmidine and spmrmine)with concentration from 30.22 ng/mg(putrescine)to 1679.98 ng/mg(spmrmidine)were found in Acyrthosiphon Pisum.In the Acyrthosiphon Pisum,all the three PAs decreased with the increase of instar.