A micro method involving micro high-performance liquid chromatography-mass spectrometry for the structural characterization of neutral glycosphingolipids and monosialogangliosides

A micro method involving high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) has been developed for the sensitive structural characterization of neutral glycosphingolipids and monosialogangliosides. The method involves a micro silica gel column (0.3 mm i.d. x 100 mm) and a micro HPLC apparatus working at a flow rate of 6 microliters/min. All injected materials can be structurally characterized by mass spectrometry without the splitting or wasting of materials, which was not possible with our previous method [M. Suzuki et al. (1990) J. Biochem. 108, 92-98]. A mixture containing 160 ng each of five neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3 alpha GalNAc-Gb4Cer) and a mixture containing 160 ng each of three monosialogangliosides [GM3(NeuAc), GM2(NeuAc), and GM1(NeuAc)] were injected into the micro HPLC with programmed elution with isopropanol-n-hexane-water with or without ammonium hydroxide. Each glycosphingolipid was separated by mass chromatography and the obtained mass spectra were suitable for structural characterization. Thus, the characterization of glycosphingolipids was achieved with small amounts of materials, 160 ng each, and in mixtures.     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1. Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model

We have shown that the ABC transporter, multiple drug resistance protein 1 (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis. Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis. In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney. The lack of renal glomerular Gb3 is retained, but the number of verotoxin 1 (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice. Adult Fabry mice were treated with alpha-galactosidase (enzyme-replacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues. Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase +/- biweekly intra peritoneal CsA. After 9 weeks, tissue Gb3 content and localization were determined using VT1/TLC overlay and histochemistry. Serum Gb3 recovered to lower levels after CsA treatment. Gb3 was undetected in wild-type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment. VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad. Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment. We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases.     

Increased globotriaosylceramide levels in a transgenic mouse expressing human alpha1,4-galactosyltransferase and a mouse model for treating Fabry disease

Fabry disease is a lysosomal storage disorder caused by an α-galactosidase A (α-Gal A) deficiency and resulting in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A transgenic mouse expressing the human α-Gal A R301Q mutant in an α-Gal A-knockout background (TgM/KO) should be useful for studying active-site-specific chaperone (ASSC) therapy for Fabry disease. However, the Gb3 content in the heart tissue of this mouse was too low to detect an ASSC-induced effect. To increase the Gb3 levels in mouse organs, we created transgenic mice (TgG3S) expressing human α1,4-galactosyltransferase (Gb3 synthase). High levels of Gb3 were observed in all major organs of the TgG3S mouse. A TgG3S (+/-)M(+/-)/KO mouse was prepared by cross-breeding the TgG3S and TgM/KO mice and the Gb3 content in the heart of the TgG3S(+/-)M(+/-)/KO mouse was 1.4 μg/mg protein, higher than in the TgM(+/-)/KO (<0.1 μg/mg protein). Treatment with an ASSC, 1-deoxygalactonojirimycin, caused a marked induction of α-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(+/-) M(+/-)/KO mouse organs. These data indicated that the TgG3S(+/-) M(+/-)/KO mouse was suitable for studying ASSC therapy for Fabry disease, and that the TgG3S mouse would be useful for studying the effect of high Gb3 levels in mouse organs.     

Fast estimation of ATP/ADP ratio as a special step in pharmacological and toxicological studies using the cell-free translation systems

We have developed a simple and effective reversed-phase HPLC procedure for rapid estimation of the ATP/ADP ratio in a cell-free translation system containing creatine kinase. Analysis of the acetone-extractable pool derived from a reticulocyte lysate cell-free system was carried out by automatic chromatography on S5CN-ODS stationary phase using a linear 10-65% pyridine elution gradient formed on the basis of methanol/water (9:1, v/v) mobile phase. This method was used to detect and characterize the inhibition of translation induced by considerable suppression of ATP resynthesis in vitro. It was shown that methyl mercury, unlike cycloheximide, pactamycin, CCl4 and barbituric acid, exerts inhibitory effect on the ATP regeneration in a cell-free translation system.     

Menstrual cycle-associated expression of 2-hydroxy fatty acyl phytosphingosine-containing GlcCer, LacCer and Gb3Cer in human uterine endometrium.

In the previous study, we found that sulfatide was characteristically expressed in the secretory phase of human uterine endometrium and that the metabolism of glycosphingolipids was strictly controlled by sex steroid hormones. Therefore, the neutral glycosphingolipid composition of human uterine endometrium in the proliferative and secretory phases was analyzed and was found to be characteristic in both phases. The major neutral glycolipids were GlcCer, LacCer, Gb3Cer and Gb4Cer. The concentrations of GlcCer, LacCer and Gb3Cer in the secretory phase were higher than those in the proliferative phase. Furthermore, on TLC, GlcCer, LacCer and Gb3Cer in the proliferative phase gave three bands, the 3rd band, which migrated to the lowest position, being much more predominant in the secretory phase. The individual band materials in both phases were purified by silica gel column chromatography, and their structures were analyzed by FABMS and GLC. The lower-migrating bands of GlcCer, LacCer and Gb3Cer were found to contain molecules with 2-hydroxy fatty acyl phytosphingosine, indicating that hydroxylation of the fatty acid and sphingosine moieties to give 2-hydroxy fatty acid- and phytosphingosine-containing glycosphingolipids, respectively, is induced selectively in the secretory phase on a change in the hormonal environment.     

Sex differences of urinary and kidney globotriaosylceramide and lyso-globotriaosylceramide in Fabry mice

The aim of our study was to measure globotriaosylceramide (Gb(3)) and lyso-Gb(3) levels by tandem mass spectrometry in the urine and kidney in Fabry (gla knockout) mice and wild-type controls. We found that urine Gb(3) of male and female Fabry mice was higher than wild-type mice of the same sex but also significantly higher in male mice compared with females of the same genotype. In kidney tissue, sex and genotype-dependent differences in Gb(3) levels paralleled those in the urine. Isoforms C16, C22:1, and C24OHA were particularly higher in males compared with females in both wild-type and Fabry mice. Similarly, kidney lyso-Gb(3) concentrations were significantly higher in 12-month-old male Fabry mice than in their homozygous female counterparts. However, lyso-Gb(3) was undetectable in wild-type mice of both sexes. α-Galactosidase A activity and mRNA levels in kidney were significantly lower in male wild-type mice compared with female mice. This study shows the sex differences in kidney and urine Gb(3) and kidney lyso-Gb(3) levels in both wild-type and Fabry mice, and it suggests that these male-female differences should be taken into consideration when using murine models for Fabry disease.     

Structural characteristics of the ceramides of neutral glycosphingolipids in the human female genital tract--their menstrual cycle-associated change in the cervical epithelium and uterine endometrium, and their dissociation in the mucosa of the fallopian tube with the menstrual cycle.

In human cervical epithelium, uterine endometrium, and mucosa of the fallopian tubes, neutral glycosphingolipids were exclusively represented by the globo-series glycosphingolipids, such as CMH, LacCer, Gb3Cer and Gb4Cer, but the molecular species of their ceramide moieties were characteristically altered in the cervical epithelium and uterine endometrium during the menstrual cycle. Individual neutral glycosphingolipids in the cervical epithelium and the uterine endometrium at the follicular phase gave two bands on TLC, whereas those at the luteal phase displayed three bands, the third being the lower migrating one. Neutral glycosphingolipids migrating to the same positions as these lower-migrating bands were constantly detected in the mucosa of the fallopian tubes, independent of the menstrual cycle. The lower-migrating bands for the cervical epithelium and the uterine endometrium at the luteal phase were due to molecules mainly constructed of phytosphingosine with alpha-hydroxy fatty acids having chain lengths of 18-24 and 4-sphingenine with alpha-hydroxy fatty acids having chain lengths of 16-22, whereas those in the mucosa of the fallopian tubes were exclusively N-alpha-hydroxypalmitoyl 4-sphingenine.     

Local and global cerebral blood flow and glucose utilization in the α-galactosidase A knockout mouse model of Fabry disease

Fabry disease is an X-linked lysosomal disorder characterized by deficient alpha-galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMR(glc)) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the alpha-galactosidase A gene knockout mouse. Global CMR(glc) was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMR(glc) ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate-putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.     

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm.Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

Distributions of Globotriaosylceramide Isoforms,and Globotriaosylsphingosine and Its Analogues in an α-Galactosidase A Knockout Mouse,a Model of Fabry Disease

Fabry disease is caused by deficient activity of α-galactosidase A (GLA) and characterized by systemic accumulation of glycosphingolipids, substrates of the enzyme. To gain insight into the pathogenesis of Fabry disease based on accumulated substrates, we examined the tissue and plasma distributions of globotriaosylceramide (Gb3) isoforms, and globotriaosylsphingosine (lyso-Gb3) and its analogues in a GLA knockout mouse, a model of Fabry disease, by means of liquid chromatography-mass spectrometry and nano-liquid chromatography-tandem mass spectrometry, respectively. The results revealed that the contents of these substrates in the liver, kidneys, heart, and plasma of GLA knockout mice were apparently higher than in those of wild-type ones, and organ specificity in the accumulation of Gb3 isoforms was found. Especially in the kidneys, accumulation of a large amount of Gb3 isoforms including hydroxylated residues was found. In the GLA knockout mice, the proportion of hydrophobic Gb3 isoforms was apparently higher than that in the wild-type mice. On the other hand, hydrophilic residues were abundant in plasma. Unlike that of Gb3, the concentration of lyso-Gb3 was high in the liver, and the lyso-Gb3/Gb3 ratio in plasma was significantly higher than those in the organs. The concentration of lyso-Gb3 was apparently higher than those of its analogues in the organs and plasma from both the GLA knockout and wild-type mice. This information will be useful for elucidating the basis of Fabry disease.     

Caveolin-associated accumulation of globotriaosylceramide in the vascular endothelium of alpha-galactosidase A null mice

Cardiovascular complications, including stroke and myocardial infarction, result in premature mortality in patients with Fabry disease, an X-linked deficiency of alpha-galactosidase A (alpha-Gal A). The enzymatic defect results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. To better understand the underlying pathogenesis of Fabry disease, the caveolar lipid content of primary cultured mouse aortic endothelial cells isolated from alpha-Gal A null mice was measured. Lipid mass analysis revealed that the excessive Gb3 in cultured alpha-Gal A-deficient mouse aortic endothelial cells accumulated in endothelial plasma membrane caveolar fractions. The levels of glucosylceramide and lactosylceramide increased in parallel with Gb3 levels in an age-dependent manner, whereas globotetraosylceramide (Gb4) levels reached maximal levels by 6 months of age and then rapidly decreased at older ages. The levels of cholesterol enriched in caveolar membranes declined in parallel with the progressive deposition of Gb3. Depleting Gb3 with recombinant human alpha-Gal A protein or d-threo-ethylenedioxyphenyl-P4, an inhibitor of glucosylceramide synthase, restored cholesterol in cultured alpha-Gal A-deficient mouse aortic endothelial cell caveolae. By contrast, recombinant human alpha-Gal A was less effective in normalizing the cholesterol content. These results demonstrate the caveolar accumulation of glycosphingolipids in an in vitro model of a lysosomal storage disease and raise the possibility that dynamic changes in the composition of plasma membrane lipid microdomains may mediate the endothelial dysfunction seen in Fabry disease.     

Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.

To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.     

Determination of ebrotidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography

Ebrotidine is a new H₂-receptor antagonist with powerful antisecretory activity, demonstrated gastroprotection and the ability to inhibit protease and lipase activities of Helicobacter pylori. As a tool in the clinical pharmacokinetic study of ebrotidine, an analytical method for the simultaneous determination of ebrotidine an its metabolites in human urine was developed. An ion-pair reversed-phase HPLC separation using 1-hexanesulfonic acid and acetonitrile as mobile phase with gradient elution was optimized. In addition, several procedures of preconcentration and clean-up were tested, including solid-phase and liquid—liquid extraction, the mixture dichloromethane—2-propanol (9:1, v/v) at pH 11 being the most efficient. The quality parameters of the whole analytical method were established, the calibration curves were linear over the range studied (1–200 μg/ml) and the reproducibility of the method was high (inter-day R.S.D. values lower than 4.4%).The limits of detection were between 26 and 110 ng/ml of urine for ebrotidine and its metabolites. The method was applied to the analysis of urine collected from two volunteers during 96 h following oral administration of ebrotidine at a dose of 400 mg.     

Identification of the neutral glycosphingolipids of murine mast cells: expression of Forssman glycolipid by the serosal but not the bone marrow-derived subclass

The expression of neutral glycosphingolipids by mouse T cell-dependent, bone marrow-derived mast cells (BMMC) obtained in vitro was determined by chromatographic and immunochemical criteria. Neutral glycosphingolipids were isolated from BMMC by extraction of 3 to 5 X 10(8) cells in chloroform/methanol (1/1, v/v) and chromatography on DEAE-Sephadex, and were analyzed by thin layer chromatography with orcinol staining. The predominant neutral glycosphingolipids of BMMC were glucosylceramide (CMH), lactosylceramide (CDH), globotriosylceramide (CTH), globotetraosylceramide (globoside), and a molecule migrating slightly faster than gangliotetraosylceramide (asialo GM1) and slower than globopentaosylceramide (Forssman glycolipid). The profiles on thin layer chromatograms of the neutral glycosphingolipids were the same for BMMC derived from BALB/c, C57BL/6, WBBF1-W/Wv, and WBBF1-+/+ mice, and for cells differentiated in either WEHI-3 conditioned medium or concanavalin A-splenocyte conditioned medium. High performance liquid chromatography of benzoylated neutral glycosphingolipids of BMMC on a Zipax column confirmed the identity of the four neutral glycosphingolipids identified by thin layer chromatography. The fifth major glycosphingolipid had an elution time greater than that of globotetraosylceramide and did not co-elute with any of the standards tested. Direct biochemical analyses of the neutral glycosphingolipids of mouse serosal mast cells (SMC) were not feasible because only 2 X 10(6) SMC could be isolated per 100 mice. However, mouse SMC bound a rat monoclonal anti-globopentaosylceramide antibody (M1/87.27.7) and rat monoclonal B1.1 antibody, as assessed by indirect immunofluorescence and flow cytometry, whereas mouse BMMC did not. The binding of B1.1 antibody to SMC could be blocked by the anti-globopentaosylceramide antibody, and the specificity of B1.1 antibody for globopentaosylceramide was confirmed immunochemically with the use of a solid phase radioimmunoassay. As estimated immunochemically, the amount of globopentaosylceramide in mouse SMC was 62 ng/10(6) cells, whereas BMMC contained less than 8 ng/10(6) cells. Thus, the expression of globopentaosylceramide is a characteristic of the mouse SMC that is lacking in the T cell-dependent BMMC.     

High-performance liquid chromatographic determination of zinc protoporphyrin and porphyrin carboxylic acids in urine

A method for the reversed-phase high-performance liquid chromatographic separation of zinc protoporphyrin and porphyrin carboxylic acids with fluorescence detection and its application are described. A mu Bondapak C18 column was employed for all the experiments in this study. The method required a pretreatment of the column with a two-component mobile phase containing 0.1 M NaH2PO4 in acetonitrile (28:15, v/v, pH 5.3) for 10 min prior to sample injection. Separation was achieved isocratically by increasing the concentration of acetonitrile in the mobile phase (0.1 M NaH2PO4:acetonitrile, 18:130, v/v, pH 5.3) 4 min after injection to complete the elution. The flow rates and the period of pretreatment of the column were studied to optimize the separation. The method was applied to determining zinc protoporphyrin and porphyrin carboxylic acids of heme biosynthesis in urine.     

Simultaneous extraction and separation of liquiritin,glycyrrhizic acid,and glabridin from licorice root with analytical and preparative chromatography

Simultaneous extraction and separation of liquiritin, glycyrrhizic acid, and glabridin from licorice were developed by liquidliquid extraction with liquid chromatography separation. By utilizing different extraction solvents, procedures, and times, the optimum extraction conditions were established. The extracts of licorice were separated and determined using a C₁₈ column with a mobile phase consisting of acetonitrile-water (containing 1.0% acetic acid) with a gradient elution of 0∼10 min from 20:80 to 60:40 (v/v). Preparative columns with different packing sizes were investigated to isolate the three compounds from the extracts of licorice. The 12 μm chromatographic column showed better separation for the three compounds from licorice. 0.29 mg/g for liquiritin, 1.43 mg/g for glycyrrhizic acid, and 0.07 mg/g for glabridin were obtained and the recoveries were 80.8, 89.7, and 72.5%, respectively.     

Anderson-Fabry disease in heart failure

Anderson-Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficiency of the enzyme alpha-galactosidase A. The worldwide incidence of Fabry’s disease is reported to be in the range of 1 in 40,000–117,000, although this value may be a significant underestimate given under recognition of symptoms and delayed or missed diagnosis. Deficiency in alpha-galactosidase A causes an accumulation of neutral glycosphingolipids such as globotriaosylceramide (Gb3) in lysosomes within various tissues including the vascular endothelium, kidneys, heart, eyes, skin and nervous system. Gb3 accumulation induces pathology via the release of pro-inflammatory cytokines, growth-promoting factors and by oxidative stress, resulting in myocardial extracellular matrix remodelling, left ventricular hypertrophy (LVH), vascular dysfunction and interstitial fibrosis. Cardiac involvement manifesting as ventricular hypertrophy, systolic and diastolic dysfunction, valvular abnormalities and conduction tissue disease is common in AFD and is associated with considerable cardiovascular morbidity and mortality from heart failure, sudden cardiac death and stroke-related death.     

LC-MS/MS 检测癫痫患者神经递质类氨基酸

目的:建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价.方法:选用AAA-C18柱色谱柱,以乙腈水(含有0.01%七氟丁酸、0.1%甲酸)为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测.疾病组与健康组的统计采用t检验和主成份分析.结果:疾病组和健康组氨基酸测定结果显示:Trp、GABA两组间没有显著性差异(P＞0.05),Arg、Glv、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异(P＜0.05),通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸.结论:试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价.