Simple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid

doi:10.1016/0378-4347(94)80085-5

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 657, Issue 1, 1 July 1994, Pages 185-191

https://doi.org/10.1016/0378-4347(94)80085-5 Get rights and content

Abstract

The quantitation of 16 acidic, basic, small and large neutral amino acids was performed using 10-μl sample aliquots of cisternal cerebrospinal fluid (CSF) and blood plasma of rats. The analytical technique is based upon a two-buffer HPLC system with fluorimetric detection of pre-column derivatized primary amino acids with o-phthaldialdehyde (OPA). A modification of a well established method, the power of the present technique comes from an improved resolution and sensitivity by installing a column heater adjusted to 43°C and strictly reducing any contamination by background amino acids. The analysis is simplified by separating the amino acid derivatives with a linear buffer gradient and less time consuming by the use of a short analytical column with a higher flow-rate. Analytical precision, linearity of response and reproducibility were highly acceptable at both CSF and plasma concentrations of amino acids without changing any of the separation or detection parameters.

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Image Pro 4.5 was used to analyze the results. At 12 h post-TBI, the glutamate level in CSF was assayed by high-performance liquid chromatography (HPLC, Beckman Company, Berkeley, CA, USA) as previously reported [Begley et al., 1994]. CSF separation was achieved on a C18 column (Supelcosil, USA) with the mobile phase composed of a mixture of methanol, sodium acetate buffer (pH 6.5; 0.1 M), and tetrahydrofuran (95:900: 5, v/v/v), at a flow rate of 1.0 mL/min and detected by a 157 spectrofluorometric detector (Beckman Company) with the excitation wavelength set at 330 nm and the emission wavelength was fixed at 450 nm.
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Short communicationSimple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid

Abstract

Prog. Brain Res.

Comp. Biochem. Physiol.

Trends Neurosci.

Brain Res. Rev.

Brain Res.

J. Neurosci. Methods

Biochim. Biophys. Acta

Anal. Biochem.

Brain Res.

J. Neurosci.

Nature

Short communication
Simple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid