Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis
Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis
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DOI：
10.1016/s0021-9673(98)00836-x
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发表时间：
1998-12-18
影响因子：
4.1
通讯作者：
Cohen, SA
中科院分区：
文献类型：
--
作者：
Liu, HJ;Sañuda-Peña, MC;Cohen, SA
本文报道了一种灵敏的测定脑和脑脊液微透析液中神经递质氨基酸（如天冬氨酸、谷氨酸和γ-氨基丁酸）亚微摩尔水平的方法。以6-氨基喹啉基-N-羟基琥珀酰亚胺基氨基甲酸酯（AQC）为衍生试剂，采用高效液相色谱法分离，荧光检测。通过将AQC直接与微透析样品混合来简单地进行衍生化。在室温下混合后，反应在数秒内完成。分离开发优化的梯度配置文件，洗脱液的pH值和柱温导致在不到30分钟的时间内所需的氨基酸在一个很好的分离。在相同的配置文件中的其他解决的氨基酸包括甘氨酸，牛磺酸，和Pro。掺入高盐灌注缓冲液中的目标氨基酸的回收率大于97%。通过在检测器中采用16 μ l流动池并分析20 μ l衍生混合物等分试样，增加了该方法的灵敏度。在优化的条件下，检测限为3-7 nM（fmol/μ l）。典型重现性（%R.S.D.）用于在亚微摩尔水平下定量这些氨基酸的浓度约为2%。在0.2-20 μ M范围内，线性良好（r（2）>0.999）。低检测限允许分析许多不同的微透析液样品，包括来自脑脊液的样品，以及来自脑样品的黑质和下丘脑，即使在γ-氨基丁酸浓度可能不高的基础水平。
A sensitive method for quantitatively determining submicromolar levels of neurotransmitter amino acids (e.g. Asp, Glu and gamma-aminobutyric acid) in microdialysates from brain and cerebrospinal fluids is reported. 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was employed as the derivatization reagent, followed by HPLC separation and fluorescence detection of the derivatives. The derivatization was conducted simply by mixing the AQC directly with the microdialysis samples. The reaction was complete within seconds after mixing at room temperature. Separation development optimizing the gradient profile, eluent pH and column temperature resulted in an excellent separation of the required amino acids in less than 30 min. Other resolved amino acids in the same profile include Gly, taurine, and Pro. Recoveries for the amino acids of interest spiked into high salt containing perfusion buffers were greater than 97%. The sensitivity of the method was increased by employing a 16-mu l flow cell in the detector and analyzing 20-mu l aliquots of the derivatization mixtures. With the optimized conditions, the detection limits were 3-7 nM (fmol/mu l). Typical reproducibility (%R.S.D.) for quantitation of these amino acids at submicromolar levels was approximately 2%. Excellent linearity (r(2)>0.999) was achieved over the range 0.2-20 mu M. The low detection limits permitted the analysis of a number of different microdialysate samples including those from cerebrospinal fluid, as well as substantia nigra and hypothalamus from brain samples, even at basal levels when gamma-aminobutyric acid concentration may be