Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry

The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI-MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, β-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5-75 fmol) were as much as 2-60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r² = 0.996-0.999, range = 100-8500 nmol L⁻¹). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC-MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.     

A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids

This study compares the main commercial detectors that can detect amino acids in their underivatized form. The detectors tested are: the chemiluminescent nitrogen detector (CLND), the evaporative light scattering detector (ELSD), the nuclear magnetic resonance spectrometer, conductivity detector, refractive index, UV, and electrospray quadrupole mass spectrometry (in simple and tandem MS mode). As ELSD, CLND and MS require a volatile mobile phase, an ion-pair reversed-phase liquid chromatographic system was selected, consisting of an octadecyl column and an aqueous mobile phase containing pentadecafluorooctanoic acid as volatile ion-pairing reagent. Underivatized taurine, hypotaurine, aspartic acid, hydroxyproline, asparagine, serine, glycine, glutamine, cysteine, glutamic acid, threonine and alanine were simultaneously analysed with each detector. In order to test the applicability of these detectors to "real world" samples, the amino acid stoichiometry of the tetrapeptide Gly-Gly-Asp-Ala was determined with each detector after acid hydrolysis. The detectors were compared in terms of linearity, limit of detection, advantages and disadvantages as well as special features (capacity to provide structural information, specificity, quantification with single calibration curve, etc.).     

Microfluidics in amino acid analysis

Microfluidic devices have been widely used to derivatize, separate, and detect amino acids employing many different strategies. Virtually zero-dead volume interconnections and fast mass transfer in small volume microchannels enable dramatic increases in on-chip derivatization reaction speed, while only minute amounts of sample and reagent are needed. Due to short channel path, fast subsecond separations can be carried out. With sophisticated miniaturized detectors, the whole analytical process can be integrated on one platform. This article reviews developments of lab-on-chip technology in amino acid analysis, it shows important design features such as sample preconcentration, precolumn and postcolumn amino acid derivatization, and unlabeled and labeled amino acid detection with focus on advanced designs. The review also describes important biomedical and space exploration applications of amino acid analysis on microfluidic devices.     

Advances in amino acid analysis

Amino acids are important targets for metabolic profiling. For decades, amino acid analysis has been accomplished by either cation-exchange or reversed-phase liquid chromatography coupled to UV absorbance or fluorescence detection of pre-column or post-column-derivatized amino acids. Recent years have seen great progress in the development of direct-infusion or hyphenated mass spectrometry in the analysis of free amino acids in physiological fluids, because mass spectrometry not only matches optical detection in sensitivity, but also offers superior selectivity. The advent of cryo-probes has also brought NMR spectroscopy within the detection limits required for the analysis of free amino acids. But there is still room for further improvement, including expansion of the analyte spectrum, reduction of sample preparation and analysis time, automation, and synthesis of affordable isotope standards. Figure Fully automated gas chromatography-mass spectrometry analysis of amino acids.     

Home-made capillary array electrophoresis for high-throughput amino acid analysis

A capillary array electrophoresis (CAE) with confocal rotary scanner for high-throughput carboxytetramethylrhodamine succinimidyl ester (TAMRA)-labeled amino acid (AA) analysis is presented. Performance of the CAE setup was evaluated with AA samples. Up to 128 capillaries could be detected in parallel. For the first time, the device was applied to separate the enantiomers of isoleucine, cysteine, and glutamic acid with cyclodextrin-modified electrolytes by capillary zone electrophoresis. Baseline separation of seven AAs is also demonstrated using micellar electrokinetic chromatography method.     

Recent advances in microchip electrophoresis for amino acid analysis

With the maturation of microfluidic technologies, microchip electrophoresis has been widely employed for amino acid analysis owing to its advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. In this article, we review the recent progress in amino acid analysis using microchip electrophoresis during the period from 2007 to 2012. Innovations in microchip materials, surface modification, sample introduction, microchip electrophoresis, and detection methods are documented, as well as nascent applications of amino acid analysis in single-cell analysis, microdialysis sampling, food analysis, and extraterrestrial exploration. Without doubt, more applications of microchip electrophoresis in amino acid analysis may be expected soon.     

Comparison of liquid chromatography with fluorescence detection to liquid chromatography-mass spectrometry for amino acid analysis with derivatisation by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate: Applications for analysis of amino acids in skin

The analysis of nineteen amino acids found in collagen was optimised using 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) as a derivatisation reagent. The analysis and detection of nineteen AQC-amino acids using fluorescence and mass spectrometry were compared at different mobile phase pH’s and column temperatures. The pH range of the mobile phase was set between 2.7 and 6.0 and column temperatures, 15–60 °C. The majority of amino acids produced a mono-derivatised product with AQC, except cystine, lysine and hydroxylysine which were di-derivatised. Hydroxylysine’s retention time was affected most by changes in the pH, whilst hydroxyproline’s retention time was more affected by column temperature. Hydroxylysine was detected as two diastereomers which were completely resolved. The relative standard deviation of the retention times of AQC-amino acids was less than 1% and the limit of detection (LOD) and limit of quantitation (LOQ) were ranged from (0.05–0.23) µM and (0.07–0.76) µM on fluorescence and (0.02–0.10) µM and (0.06–0.33) µM on mass spectrometry respectively. This method was successfully applied for the quantitation of amino acids in different animal skins.     

Comparison of portable detectors for uranium enrichment measurements

Uranium enrichment and holdup measurements require a detector capable of accurately obtaining the 186-keV peak area. NaI detectors have been widely used for these tasks. However, for recycled uranium, the interference of the 239-keV peak from the ²³²U decay chain challenges the capabilities of the NaI detectors to accurately extract the area of the 186-keV peak. Using CZT detectors, which have much better resolution than the NaI detectors, has temporarily solved this interference problem. However, the CZT detectors have setbacks in that they are generally small and have low efficiencies, which require long acquisition times for reasonable statistics. Recently, two new types of scintillator detectors have become available commercially, LaCl₃(Ce) and LaBr₃(Ce). These cerium-doped lanthanum halide detectors, with comparable resolution but better efficiency than the CZT detectors, appear to permanently solve the interference problem for recycled uranium measurements. In this report, we compare the uranium enrichment measurement performances of a portable NaI detector, a large coplanar-grid CZT detector, and a LaBr₃ detector.     

Comparison of resolving time values of different scintillation detectors in the coincidence experiments

The resolving time values of the coincidence spectrometers composed by NaI(Tl) inorganic, BC408 type and BC418 type organic scintillation detectors were obtained in this work. For this purpose, fast–fast coincidence measurements were carried out. Best timing performance was obtained by BC418 type plastic scintillator for various source-detector distances.     

Comparison of monomeric and polymeric amino acid based surfactants for chiral separations

To better understand chiral recognition with polymeric amino acid based surfactants, the chromatographic performance of 18 monomeric and polymeric surfactants were compared for chiral analytes with various charge states and hydrophobicities. In this study, four amino acids (glycine, L-alanine, L-valine, and L-leucine) were chosen, and all possible combinations of the chiral single amino acid and dipeptide surfactants were synthesized. The results indicate that polymeric surfactants usually provide better chiral resolution for enantiomers of lorazepam, temazepam, 1,1'-bi-2-naphthol, and propranolol as compared to monomeric surfactants. In contrast, monomers perform better for chiral recognition of the 1,1'-bi-2-naphthyl-2,2'-diyl hydrogenphosphate enantiomers.     

Comparison of amino acid sequence analysis by electron ionization and negative-ion chemical ionization mass spectrometry

The positive electron ionization and negative chemical ionization mass spectra of 15 different derivatives of the tripeptide Phe-Ala-Leu have been compared. Total ion currents and ion currents of sequence-characterizing ions have been measured and compared. The negative-ion spectra, using 10% carbon dioxide in argon as moderator gas, proved to be simpler and contained more abundant sequence ions than the positive electron ionization spectra.     

Computer analysis of amino acid chromatograms.

A procedure is described for the automatic off-line analysis of amino acid chromatograms of protein hydrolysates, using a small computer. The data requirements are basic, and, unlike previous programs, the present system allows the separation and identification of bands, as well as the quantitative determination of composition. With minor modification, the program could be extended for use with most types of chromatographic data. The validity of the application of the program to experimental data is discussed.