生物样品中游离氨基酸的测定

生物样品,包括生理体液,如血液、淋巴、尿、脑脊髓液等和生物组织,如动物的脏器、肌肉、植物的根、茎、叶、花、果实等等。其中游离氨基酸含量的测定,对研究动植物体内代谢,临床诊断,营养水平的分析都有一定的帮助。因而生物样品中游离氨基酸的测定越来越显得重要。我们用磺基水杨酸沉淀法,脱去组织及体液中的蛋白     

阴离子交换色谱-积分脉冲安培检测法测定寿胎丸中游离氨基酸

建立了阴离子交换色谱-积分脉冲安培检测法测定寿胎丸煎煮液中游离氨基酸的分析方法。实验考察了18种氨基酸的梯度洗脱条件,优化了固相萃取条件,最终选用SSDBX型固相萃取小柱净化煎煮液。在优化实验条件下,进行了加标回收实验,回收率在71.2%~120.9%之间,相对标准偏差为0.8%~10.4%。18种氨基酸的检测限为0.07~5.00μg/L。方法能满足寿胎丸煎煮液中氨基酸的检测要求。     

阴离子交换色谱-积分脉冲安培法测定鱼粉和玉米粉水解液中氨基酸

将高效阴离子交换色谱．积分脉冲安培直接检测法应用于鱼粉和玉米粉水解液中的氨基酸的分析测定。该方法使用Dionex AminoPac PA10阴离子交换柱为分析柱，控制柱温为35℃，以NaOH和NaAc的强碱性溶液为淋洗液，在合适的梯度条件下，可以实现对17种常见氨基酸的高效分离。在强碱性条件下，获得良好分离的各个氨基酸可在配有金工作电极的电化学检测器上以积分脉冲安培法获得高灵敏度的检测。在优化的测定条件下，该方法对这17种氨基酸的检出限在0．21—3．38pmol之间；峰面积校正曲线的线性范围为2—3个数量级；5次平行测定的峰面积的相对标准偏差RSD在0．32％—4．7％之间。该方法应用于玉米粉和鱼粉水解液中氨基酸含量的测定，结果良好。对玉米粉和鱼粉水解液标准加入的回收率分别在81．9％—108．0％和90．0％～108．0％之间。     

蛋白质水解液中氨基酸组成的定量分析

以氯甲酸－9－芴基甲酯（FMOC－Cl）为柱前衍生化试剂在液相色谱上分离17种常见氨基酸的衍生物并测定其摩尔吸光系数。用绝对分析法和标准曲线法对标准氨基酸溶液进行了定量分析，除组氨酸外两种方法测定结果的相对偏差为5．00％以内。对氨基酸的稳定性进行了模拟水解实验，发现在110℃/24h的水解条件下，丝氨酸等不稳定氨基酸的降解较在150℃/1.5h条件下小。用绝对分析法测定测定了牛胰岛素不同水解条件下水解液中氨基酸的含量，大部分氨基酸的相对收率都接近100％。     

急性力竭运动对大鼠下丘脑氨基酸神经递质的影响

用激光诱导荧光检测的高效毛细管电泳结合微透析技术测定急性力竭运动前后的大鼠下丘脑区细胞外液中的氨基酸神经递质的含量。实验结果表明，急性力竭运动后大鼠下丘脑区的细胞外液中的6种氨基酸（精氨酸、甘氨酸、赖氨酸、谷氨酸、丙氨酸、γ－氨基丁酸）有统计意义的增加，这一结果显示了下丘脑区的氨基酸在应激过程中很可能起着重要的作用。     

碳糊电极吸附伏安法测定痕量锑的研究

利用液体石蜡作粘合剂制成ＣＰＥ，根据锑与茜素络合剂生成的络合物在ＣＰＥ上的吸附特性，考察了最佳底液条件，共存离子的影响，测定了电活性络合物的组成，探讨了电极反应机理。方法已用于水样中锑的测定，检出限为４＊１０＾－９ｍｏｌ／Ｌ。     

氨基酸手性色谱分离

光学活性和立体构象不同的氨基酸，具有不同的生理活性和作用，因此，实现氨基酸的有效手性分离具有重要意义。色谱法是常用的氨基酸手性分离方法，具有分离效率高、速度快、灵敏、成本低和绿色环保等特点，在氨基酸手性分离和检测领域应用广泛。本文综述了色谱法在氨基酸手性分离方面的最新进展，并对其发展趋势进行了展望。     

硅酸铜分离微生物发酵液中的氨基酸

采用硅酸铜对微生物发酵液中的氨基酸进行了分离研究.通过静态吸附试验考察了硅酸锌和硅酸铜对微生物发酵液中氨基酸的吸附影响.结果表明,硅酸铜比硅酸锌更适合分离发酵液中的氨基酸,并确定了以硅酸铜为填料的分离条件:上柱发酵液pH=2,流速0.2BV/h,洗脱液为1mol/L的NH4Cl.在该条件下能够一次性将3种不同的氨基酸较好的分离.     

相转移催化不对称合成(Ⅰ)——相转移催化反应合成光学活性α-氨基酸

在固液两相条件下用手性季铵盐为相转移催化剂,邻苯二甲酰亚胺钾与α-溴代羧酸酯反应,合成了十二个具有光学活性的α-(邻苯二甲酰亚胺基)羧酸酯,经肼解和水解,得到相应的光学活性α-氨基酸,光学纯度百分率为3.6—17.8％。     

氨基酸荧光分析法应用

在海洋化学的许多研究中，经常涉及到海水介质、大批量样品中氨基酸的快速灵敏的分析。此文就荧光分析法直接测定氨基酸的分析条件及影响因素等进行了研究。     

Measurement of total plasma and cerebrospinal fluid homocysteine by fluorescence following high-performance liquid chromatography and precolumn derivatization with o-phthaldialdehyde.

Precolumn derivatization of amino acids with o-phthaldialdehyde followed by high-performance liquid chromatographic separation and fluorescence detection is used in many clinical and experimental laboratories for the measurement of primary amino acids. This technique was adapted for the measurement of homocysteine in plasma and cerebrospinal fluid (CSF) following alkylation of the free sulphydryl group with iodoacetate. The minimum detection limits are less than 1 microM in plasma and 80 nM in CSF. Within-day and between-day coefficients of variation for plasma and for CSF are less than 10%. Values for normal plasma homocysteine range from 6.04 to 16.2 microM and for CSF from 0.28 to 0.66 microM.     

Amino acid analysis of physiological fluids by high-performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography

The suitability of pre-column derivatization with phenylisothiocyanate followed by high-performance liquid chromatography was investigated as a means of analyzing free amino acids in plasma and other physiological fluids. A comparison was made between this method and a conventional ion-exchange method. The correlation coefficient for all the amino acids tested was greater than 0.9, except for proline and tryptophan. Various forms of sample preparation were tried for plasma and amniotic fluid; it was finally decided that protein precipitation with acetonitrile was most suitable. Ultrafiltration was used for cerebrospinal fluid preparation while urine was treated the same as a standard mixture. The retention times relative to the internal standard (nor-leucine) are given for over 90 compounds. Some of these were chromatographed underivatized because they are known to be present in some physiological fluids and absorb at 254 nm because of their aromaticity. The imprecision for this method compared favourably with the standard ion-exchange method although each had specific amino acids for which the imprecision was poor. The technique is suitable for the same routine clinical analysis purposes as high-resolution ion-exchange chromatography. It also offers the advantages of speed of analysis, sensitivity and equipment versatility over the conventional ion-exchange methods.     

Simultaneous liquid chromatographic determination of seventeen of the major monoamine neurotransmitters, precursors and metabolites. II. Assessment of human brain and cerebrospinal fluid concentrations

The optimized chromatographic method procedure presented in Part I was employed for the assessment of human brain and cerebrospinal fluid neurotransmitters levels. The optimized sample preparation and chromatographic conditions permitted a rapid (less than 25 min), sensitive and semi-automated high-performance liquid chromatographic analysis which measures all major monoamine neurotransmitters, precursors and metabolites in human brain and cerebrospinal fluid. The brain specimen was deproteinized with perchloric acid (containing Na2EDTA and sodium sulphite), the internal standard and heparin were added and the samples were sonicated, centrifuged, filtered and injected directly into the chromatographic system. Cerebrospinal fluid was handled in a similar manner except that sonication was excluded. The regional distribution of monoamine neurotransmitter concentrations in human brain and cerebrospinal fluid is presented.     

Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis.

Free amino acids were quantitatively determined in cerebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthaladehyde in the presence of 2-mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was less than 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas less than 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age-matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and gamma-aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.     

Simultaneous analysis of sulfur-containing excitatory amino acids using micellar electrokinetic chromatography with diode array and laser-induced fluorescence detection

The suitability of micellar electrokinetic chromatography (MEKC) coupled with diode array or laser-induced fluorescence (LIF) detection to analyze the four sulfur-containing excitatory amino acids (SEAA), homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA), and cysteic acid (CA) was investigated. 5-Carboxy-fluorescein succinimidyl ester was chosen as fluorescent reagent to derivatize HCSA, HCA, CSA, and CA. During method development, the yield of reaction dependent on pH and incubation time as well as the stability of the products were analyzed. The maximum yield was obtained after 30 min using a 0.1 M borate buffer (pH 8.9) as derivatization buffer. Each labeled amino acid exhibited high stability at room temperature over a period of 5 days. Baseline separation of labeled HCSA, HCA, CSA, and CA was obtained using a buffer consisting of 0.1 M borate, 50 mM sodium dodecyl sulfate (SDS), and 5% v/v methanol (pH 9.0). By applying LIF detection, limits of detection ranged from 0.9 x 10(-10) M for HCSA to 6.0 x 10(-10) M for CA, respectively. Slightly modified separation conditions enabled the analysis of SEAA in cerebrospinal fluid in the presence of the neurotransmitters glutamate and aspartate. In conclusion, MEKC coupled with LIF detection is a suitable technique for the simultaneous and sensitive analysis of SEAA. Further work will focus on the validation of the method with cerebrospinal fluid as sample matrix.     

Determination of amino acids in human cerebrospinal fluid by gas chromatography

In the present study, the use of gas chromatography (GC) for the determination of amino acids in human cerebrospinal fluid (CSF) is described. Although some amino acids may be determined using a packed column following the removal of glucose, the major interfering component, the inadequate resolution of other amino acids from remaining unidentified components results in poor quantitation. The use of wide bore columns improves reproducibility considerably, but still it does not provide sufficient resolution to enable quantitative determination of all amino acids in human CSF. Good reproducibility data, with CV values for all amino acids of 7% or less and recoveries generally between 80% and 100%, can only be obtained using the fused silica open tubular (FSOT) column. Normal amino acid levels are presented for 10 samples of human CSF, which compare well with data previously reported in the literature.     

Gradient elution isotachophoresis with direct ultraviolet absorption detection for sensitive amino acid analysis

This work demonstrates coupling of the newly described electrophoretic enrichment technique of gradient elution isotachophoresis (GEITP) to a low-cost, conventional ultraviolet absorbance detector to realize sensitive measurements with a universal detector, eliminating the need for fluorescent analytes or derivatization. The effects of various parameters on enrichment were studied, including current density varied by leading electrolyte concentration, current density varied by applied electric field, and counter-flow acceleration across varying capillary inner diameters. Optimized parameters were applied to the enrichment and separation of the amino acids tryptophan (Trp) and tyrosine (Tyr). Limits of detection for Trp and Tyr were 51 and 215 nM, respectively, reflecting sensitivity enhancements of 860- and 1900-fold. Analysis times were less than 6 min, and peak height RSDs were less than 4%. A demonstration of enrichment and separation of these amino acids from artificial cerebrospinal fluid is additionally shown as a first step to realizing biochemical monitoring by GEITP.     

Chemiluminescence determination of 20 amino acids and the mechanism study

<正>It was found that 20 amino acids could inhibit the intensity of the luminol-H_2O_2-CuSO_4 chemiluminescence system.Using this character,a rapid and sensitive method for the determination of 20 amino acids was developed with flow injection coupled with chemiluminescence detection.Under the optimal conditions,the detection limits of 20 amino acids were in the range of 4.5×10~(-7)- 4.3×10~(-10) mol/L,and the relative standard deviations were less than 3.2%.The proposed method was successfully applied to drug analysis.The possible mechanism was also discussed.     

Alterations of plasma and cerebrospinal fluid glutamate levels in rats treated with the N-methyl-D-aspartate receptor antagonist, ketamine

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia.     

A facile and sensitive chemiluminescence detection of amino acids in biological samples after capillary electrophoretic separation

It was found that native amino acids enhanced the chemiluminescence (CL) reaction between luminol and BrO(-) in an alkaline aqueous solution. This has led to the development of a facile and highly sensitive CL detection scheme for the determination of amino acids in biological samples after capillary electrophoretic (CE) separation. The CE-CL conditions were optimized. An electrophoretic buffer of 2.5 x 10(-2) M sodium borate (pH 9.4) containing 1 x 10(-4) M luminol was used. The oxidizer solution of 8 x 10(-4) M NaBrO in 0.1 M sodium carbonate buffer solution (pH 12.5) was introduced post-column. Under the optimal conditions, the detection limits were 1.0 x 10(-7) M for glutamic acid (Glu) and 1.3 x 10(-7) M (S/N = 3) for aspartic acid (Asp). The relative standard deviations (RSDs) of peak area and migration time were in the ranges of 3.8-4.3% and 1.4-1.6%, respectively. The present method was applied to the determination of excitatory amino acids (i.e., Asp and Glu) in rat brain tissue and monkey plasma. The levels of these major excitatory amino acids in monkey plasma were quantified for the first time and found to be 1.17 +/- 0.17 x 10(-5) M (mean +/- SD, n = 6) for Glu and 1.64 +/- 0.19 x 10(-6) M for Asp, which were comparable with the levels in human plasma.