| Alanine aminotransferase(ALT)is an important amino acid metabolizing enzyme in the silkworm(Bombyx mori L.),which can convert glutamic acid to alanine(low content in mulberry leaves and up to 30%in the silk),thus affecting silk protein production of silkworm.Mulberry leaves contains a variety of pharmacological components such as alkaloids and amino acids,and thus has abundant physiological activities.Fagomine is a polyhydroxylated piperidine alkaloid in mulberry leaves.Due to its hypoglycemic and other biological activities,fagomine is not only used for the treatment of diseases,but also as dietary supplements and functional foods.Gamma-aminobutyric acid(GABA)is an important component in mulberry leaves and belongs to non-protein amino acids.GABA has many physiological activities such as lowering blood pressure,treating neurodegenerative diseases and so on.Direct analysis in real time(DART)ionization source is a non-surface contact,thermal analysis and plasma ionization technique that enables in-situ,direct and rapid sample analysis in atmospheric pressure environments.As there are many improvements in detection methods,based on the advantages of DART ion source,this study aims to establish a new method(Direct analysis in real time ionization source tandem mass spectrometry,DART-MS)for the determination of silkworm tissues ALT activity,fagomine and GABA in mulberry leaves.It not only enriches the determination methods of each component,but also provides experimental basis for the in-depth study of DART ion source in silkworm and mulberry leaves.1.This study established a method(DART-MS)for the determination of ALT activity in fifth-instar silkworm tissues,and compared it with the traditional method(Reitman Frankel assay).First,the parameters that affect the analysis of substances by the DART ion source were optimized.The characterization of alanine by DART-MS was discussed:in positive ion mode,the alanine standard has two characteristic ions(m/z 90[M+H]~+and m/z 179[2M+H]~+);after the secondary mass spectrometry analysis,the ion m/z 90 only gives m/z 44.3[M+H-HCOOH]~+;in the negative ion mode,the resulting spectrum has a complex background,many impurity peaks and low response intensity;therefore,the m/z 90→m/z 44.3 was selected as the quantification ion pair and experiments was performed in positive ion mode and SRM mode.The standard curve obtained by DART-MS method was good,the precision(RSD%)was 5.10%,4.61%,and 10.90%,respectively,and the recoveries were in the range of 97.61%-112.07%.Comparing the two methods,the results of DART-MS showed a high consistency with that of the Reitman Frankel assay,indicating that the DART-MS method is a reliable method for the determination of ALT activity in silkworm tissues.2.This study established a DART-MS method to qualitatively and quantitatively analyze fagomine in mulberry leaves.The conditions of MS and DART ion source were optimized.In the positive and negative ion modes,the full scan of fagomine standard was performed.The mass spectrum showed that the spectrum in the positive ion mode has higher response intensity and no magazine peak;combined with the secondary mass spectrometry analysis,m/z 148→m/z 130 was final selected as the quantification ion pair,subsequent operations were performed in SRM mode and positive ion mode.Full scan and secondary mass spectrometry analysis of mulberry leaves extracts and fagomine standard showed that the same ion peaks can be observed in both mass spectra and have similar relative abundances.Thus,the ion m/z148 in the mulberry leaves extract wss determined to be[Fagomine+H]~+.The method validation results of DART-MS method showed that the calibration curve was good;the precision(RSD%)was 2.46%,4.17%,and 4.92%;the spike recovery was between88.75%and 98.50%.Moreover,the quantitative analysis of fagomine in 8 kinds of mulberry leaves in different regions was performed using DART-MS method.3.This study established a method for the qualitative and quantitative detection of GABA content in mulberry leaves using DART-MS.The MS and DART ion source parameters were optimized firstly.The full scan spectra of positive and negative ion modes of the GABA standard confirm that the positive ion mode is more effective;while the MS/MS spectrum shows that m/z 87 is the main fragment peak,so m/z 104→m/z 87 was selected as a quantitative ion pair,and experiments were performed in SRM mode.By comparing the full scan and the MS/MS spectrum of the mulberry extracts and the GABA standard,it was found that the two have the same fragment ions and response intensity;therefore,we determine the ions m/z 104 in the extracts is[GABA+H]~+.The method validation showed that the DART-MS method had good calibration curve,with RSD%of 3.98%,5.60%,and 2.28%and the spike recovery was within 85.60%and 97.47%.In addition,the content of GABA in 6 kinds of mulberry leaves from different regions was measured by DART-MS method.The transamination of ALT can affect the synthesis of silk protein in silkworm.The new method established by us to determine the ALT activity in silkworm tissues is of great significance for silk production and sericulture benefit.The DART-MS method is more environmentally friendly than the Reitman Frankel method.It can not only monitor the progress of the enzyme reaction in real time,but also quickly determine enzyme activity.Fagomine and GABA in mulberry leaves have been used in various fields due to their rich physiological activities.The DART-MS method established in this paper does not require a complicated and tedious sample preparation processes,which largely avoids the waste of time and resources.The method allows rapid,direct,high-throughput,and high-sensitivity analysis of samples.The content of fagomine and GABA in mulberry leaves determined by DART-MS shows that there are differences in the contents of them in mulberry leaves in different provinces.We infer that this may be related to the mulberry variety and the soil,temperature,humidity and other environmental factors in the production area. |
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