| The inherited metabolic diseases are caused by gene mutations that change thefunction of certain enzymes, carrier proteins, membranes or receptors in vivo. Aseriesof clinical symptoms will appear due to the accumulation of intermediate or bypassmetabolites or lack of terminal metabolites. Patients without timely diagnosis andtreatment will be damaged the nervous system, affect intelligence, even lead to comaor death. Therefore, it is essential to screening inherited metabolic diseases as early aspossible.Liquid chromatography tandem mass spectrometry method can be used to detectthe multiple metabolites simultaneously to screening of inherited metabolic diseaseswith high sensitivity and specificity. The characteristics of low false-positive rate andhigh-throughput make it suitable for large-scale clinical application. In this researchwe established the method of quantitative of amino acids and acyl carnitines in vivoby liquid chromatography tandem mass spectrometry. It could realize the screening ofamino acids, organic acids, fatty acids metabolic diseases simultaneously. We alsoexplored the effect of different age and region to the amino acids and acyl carnitines.The detection method of10kinds of amino acids and32kinds of acyl carnitinemetabolites were established. Amino acids and acyl carnitines were extracted fromdried blood spots using the methanol with internal standards, and then derivatized byn-butanol. Finally the samples were analyzed by liquid chromatography tandem massspectrometry. Most of α-amino acids were scanned by neutral loss mode, losing thefragment of102.1Da. Some of acyl carnitines were scanned by parent scan mode, themass to charge ratio of the daughter ion was85Da. Other amino acids and acylcarnitines were scanned by multiple reaction monitoring scan mode. The amount ofamino acids and acyl carnitines in blood was calculated by Masslynx software.This method could screen13kinds of amino acids,13kinds of organic acids and11kinds of fatty acids metabolic disease simultaneously. When the detection valueswere significant over or below the reference value that suggested the person maysuffer from such diseases. There were no abnormal persons in the205cases. Inaddition, we also used target metabolomics approach to analysis the age and regionfactors by multivariate statistical analysis. We combined partial least squaresdiscriminant analysis with T-test to identify the difference metabolites. For differentage, the concentration of the amino acids and acyl carnitines in blood were different.Compared with infants ages in30days, valine, citrulline and free carnitine levels were significant increase in age over30days, while glycine, tyrosine, alanine, leucine,acetyl carnitine, ornithine acid were decrease. The results showed age-related thatsuggested the importance of using appropriate reference values when working withdifferent age’s population. For the plateau population, the concentration of glycineand alanine were higher than of in plain population, although arginine is lower. Thesemay associate with hypoxia environment and energy metabolism. This would providethe evaluation of nutrition and health status.Peptide drugs are a class of important biological active substances, which are theimportant field in pharmaceutical research. Since the peptide drug is comprised ofamino acid mainly, the quality control method is different from chemicals drug. Inaddition to refer general chemicals drug to analyze conventional item, it should doquality control research according to the characteristics of the peptides.Exenatide, a glucagon-like peptide-1(GLP-1) analogue, is a synthetic peptidewhich containing39amino acids. It could control the glucose in patients with type Ⅱdiabetes. The exenatide can stimulate the proliferation of β cell and promote thesecretion of insulin, suppress the glucagon release and slow gastric emptying, inhibitfood intake. In the worldwide, it is the first new drug for treatment of Ⅱ diabetes inrecent years.The purpose of this study is quality control of the production process ofexenatide. According to Chinese Pharmacopoeia and guiding principles of drugpharmaceutical research techniques, combined with exenatide synthesis process andreference materials, we established the quality control method of exenatide.This research analyzed the purity, molecular weight, amino acid sequences,amino acids composition, peptide mapping, isoelectric point, IR, UV, relevantmaterial to quality control of exenatide. The purity of exenatide detected by HPLCwas99.63%. Using matrix-assisted laser desorption time of flight mass the relativemolecular weight is4186.86Da, with the theoretical molecular weight of4186.60Da.Using the method of Edman degradation and tandem mass spectrometry15N-terminal amino acid sequence and full sequence of exenatide were detected. Theresults were the same as the theoretical sequence and demonstrated there was notruncated peptide or modified peptide. The result of amino acid composition analysisdemonstrated the kind and content of the amino acid of exenatide sample wascorresponding with theoretical. The deviations of amino acids were less than1.5%.Four characteristic peptides could be detected in peptide mass fingerprinting. The isoelectric point was detected by gel electrophoresis and capillary isoelectricfocusing-whole column imaging detection (CIEF-WCID). The results were similar,but the CIEF-WCID required shorter time. UV and IR information of exenatideshown the sample contained a conjugated double, peptide bond, a phenolic hydroxyl,methyl, methylene and phenyl groups.We first established the CIEF-WCID method for analyzing the related substancesin exenatide sample. Firstly, using tandem mass spectrometry to analyze the fullsequence of related substances, truncated peptide3-39, N-terminal acetylated peptideAc-His, oxidized peptide Met14SO and truncated peptide10-39, which were arisenfrom synthesis. Secondly, we investigated the accuracy and repeatability of theCIEF-WCID method. The relative errors of three peptides were no more than2%.And the intra-and inter-day precisions were less than0.12%and0.24%, whichindicated that the method had good accuracy and repeatability. Finally, CIEF-WCIDmethod was used to separate and analyze the exenatide and related substances.Different concentrations and different types of solubilizer (urea, glycerin or sucrose),different range and brand of carrier ampholytes were tried to improve separation ofexenatide and related substances. AES pH3-10and Sigma pH4-6carrier ampholyteswere chosen to detect the sample. This result suggested that the CIEF-WCID methodcould be used for separation and quality control of peptides, which provided a newtechnique for peptides and proteins quality research in the future.
|
PDF Full Text Request