Automated determination of neuroactive acidic sulphur-containing amino acids and gamma-glutamyl peptides using liquid chromatography with fluorescence and electrochemical detection

A column liquid chromatographic method is presented for the determination of trace levels of acidic sulphur-containing amino acids and gamma-glutamyl di- and tripeptides in microdialysates sampled from rat brain in vivo. Automated precolumn derivatization was performed with o-phthaldialdehyde-beta-mercaptoethanol. The derivatives were separated by reversed-phase liquid chromatography with electrochemical and fluorescence detection. The mean relative standard deviation (n = 10) was 1.03 and 4.59% for retention times and peak heights, respectively. The mean correlation coefficient of linearity (r) was 0.9982 in the range 4.5-450 pmol (n = 15), and the lowest detectable amount was 200 fmol for the homocysteinesulphinic acid derivative, (k' = 5.4, at a signal-to-noise ratio of 3). A microcolumn electrochemical detection method, developed for volume-limited samples, produced a fifteen-fold increase in mass sensitivity. Neurochemical applications using microdialysis in vivo are presented.     

Rapid simultaneous determination of amines and organic acids in citrus using high-performance liquid chromatography

Rapid analytical method for the simultaneous separation and determination of amines and organic acids is a vital interest for quality control of citrus and their products. In the present study, a simultaneous high performance liquid chromatography (HPLC) method for the rapid separation of three amines and two organic acids was developed. Chromatographic separation of compounds was achieved using Xbridge C₁₈ column at ambient temperature, with an isocratic mobile phase of 3 mM phosphoric acid at a flow rate of 1.0 mL min⁻¹. A photodiode array (PDA) detector was used to monitor the eluent at 223 nm and 254 nm with a total analysis time of 10 min. Extraction of amines and organic acids from citrus juice was optimized. The method was validated by tests of linearity, recovery, precision and ruggedness. The limit of detection (LOD) and limit of quantification (LOQ) for amines and ascorbic acid were determined to be 5 ng and 9.8 ng, respectively. All calibration curves showed good linearity (R² ≥ 0.9999) within the test ranges. The recoveries of the amines and organic acids ranged between 84% and 117%. The identity of each peak was confirmed by mass spectral (MS) analysis. The developed method was successfully applied to analyze the content of amines and organic acids in six different species and two varieties of citrus. Results indicate that mandarin and Marrs sweet orange contain high level of amines, while pummelo and Rio Red grapefruit had high content of ascorbic acid (137-251 μg mL⁻¹) and citric acid (5-22 mg mL⁻¹). Synephrine was the major amine present in Clementine (114 μg mL⁻¹) and Marrs sweet orange (85 μg mL⁻¹). To the best of our knowledge, this is the first report on simultaneous separation and quantification of amines and organic acids in Marrs sweet orange, Meyer lemon, Nova tangerine, Clementine, Ugli tangelo and Wekiwa tangelo.     

Rapid automated high performance liquid chromatography method for simultaneous determination of amino acids and biogenic amines in wine, fruit and honey

This paper reports a new, simple, rapid and economical method for routine determination of 24 amino acids and biogenic amines in grapes and wine. No sample clean-up is required and total run time including column re-equilibration is less than 40min. Following automated in-loop automated pre-column derivatisation with an o-phthaldialdehyde, N-acetyl-l-cysteine reagent, compounds were separated on a 3mm×25cm C(18) column using a binary mobile phase. The method was validated in the range 0.25-10mg/l; repeatability was less than 3% RSD and the intermediate precision ranged from 2 to 7% RSD. The method was shown to be linear by the 'lack of fit' test and the accuracy was between 97 and 101%. The LLOQ varied between 10μg/l for aspartic and glutamic acids, ethanolamine and GABA, and 100μg/l for tyrosine, phenylalanine, putrescine and cadaverine. The method was applied to grapes, white wine, red wine, honey and three species of physalis fruit. Grapes and physalis fruit were crushed, sieved, centrifuged and diluted 1/20 and 1/100, respectively, for analysis; wines and honeys were simply diluted 10-fold. It was shown using this method that the amino acid content of grapes was strongly correlated with berry volume, moderately correlated with sugar concentration and inversely correlated with total acidity.     

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high‐throughput amino acid analysis, using precolumn derivatization high‐performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI‐MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home‐built auto‐sampler system. Amino acids were derivatized with 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate, and a 3 μm Wakosil‐II 3C8‐100HG column (100 × 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra‐ and inter‐precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequential ethoxycarbonylation, methoximation and tert-butyldimethylsilylation for simultaneous determination of amino acids and carboxylic acids by dual-column gas chromatography

Amino acids (AAs) in alkaline solution were first ethoxycarbonylated with subsequent methoximation of keto acids (KAs). After acidification and solid-phase extraction, tert-butyldimethylsilylation was performed for direct analysis by gas chromatography (GC) on dual-columns with different polarities, which provided simultaneous separation of multiple amino acids, carboxylic acids (CAs) and keto acids, facilitating accurate peak confirmation based on matching with retention index sets characteristic of each analyte. The present method was linear (r2 > or = 0.9955) with good precision (0.1-9.4%) and accuracy (-8.6 to 9.9%), allowing simultaneous screening for diagnostic amino acids along with carboxylic acids and keto acids in urine from a phenylketonuria patient.     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

The determination of steric purity of amines and amino acids by gas chromatography and mass spectrometry

Amines and amino acids have been condensed with a mixture of N-TFA-R-prolyl chloride and n-TFA-S-[1-²H]-prolyl chloride and the resulting four diastereoisomers have been separated by gas chromatography into two peaks. The amount of each diastereoisomer formed was then determined by chemical ionization mass spectrometry. The steric purity of the enantiomeric mixture can be calculated from these values. The method is not dependent on the diastereoisomer formation going to completion.     

Simultaneous determination of 23 amino acids and 7 biogenic amines in fermented food samples by liquid chromatography/quadrupole time-of-flight mass spectrometry

A novel liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS) method was developed for the simultaneous determination of 23 amino acids and 7 biogenic amines in food samples. These analytes were pre-column derivatized with dansyl chloride and then separated in an Acquity column (1.7 μm; 2.1 mm × 100 mm). The separation of 31 compounds including an internal standard was achieved within 25 min at a flow rate of 0.2 mL/min. The method linearity for each amino acid and biogenic amine had a relatively wide range with r(2)>0.99. The intra- and inter-day precision, expressed as relative standard deviation (RSD), ranged from 1.1 to 4.6% and from 2.0 to 11.2%, respectively. The limit of detection was between 0.005 and 0.4 μg/mL. With a simple dilution, recoveries of around 80-120% were obtained for most of the compounds. No significant matrix effect was observed, and the developed method was successfully applied to the analysis of amino acids and biogenic amines in beer, cheese and sausage samples.     

Rapid determination of amino acids by high-performance liquid chromatography: release of amino acids by perfused rat liver.

Perfused rat liver can be considered as one of the most suitable ex vivo models for studies of liver metabolism. To assess the possible effect of L-carnitine and some of its acyl esters on proteolysis in the rat liver, the amino acid derivatization and high-performance liquid chromatographic separation of Tapuhi et al. [Anal. Biochem., 115 (1981) 123] was modified.     

Automated precolumn derivatization device to determine neurotransmitter and other amino acids by reversed-phase high-performance liquid chromatography

A device to derivatize amino acids with o-phthaldialdehyde, which is directly connected to high-performance liquid chromatographic equipment is described. Its principle is that a sample (10-500 microliters) is mixed with a reagent (containing o-phthaldialdehyde, 2-mercaptoethanol and sodium hydrogen carbonate buffer), using a peristaltic pump. This mixture is pumped into a loop of a pneumatically controlled injection valve at atmospheric pressure. When the derivatization is complete the valve switches, so that the sample is applied to a column and the amino acid derivatives are separated with a gradient of methanol-phosphate buffers. The reproducibility is such that brain perfusates or tissue extracts can be analyzed for the amino acid transmitter content and no internal standard is necessary. The major advantages of the present device are that it produces thorough mixing of reagent and sample, so that a high and constant degree of derivatization occurs (thus producing high sensitivity; less than 0.1 pmol can be detected) and its low cost.     

Automated on-line microdialysis sampling coupled with high-performance liquid chromatography for simultaneous determination of malondialdehyde and ofloxacin in whole blood

We have developed a system that couples an on-line microdialysis (MD) system with flow injection high-performance liquid chromatography (HPLC)-fluorescence detection for simultaneous measurement of the concentrations of malondialdehyde (MDA) and ofloxacin (OFL) in whole blood samples. The sample matrix was first cleaned with an MD system using an MD probe. A continuously flowing dialysate stream was derivatized on-line and auto-injected into a separation column. MDA and OFL were separated through a reverse-phase C18 column (250 mm × 4.6 mm) at a flow rate of 0.8 mL min⁻¹ and then detected using a fluorescence detector (excitation: 532 nm; emission: 553 nm); the system's components were connected on-line using a valve control. Validation experiments demonstrated good linearity, precision, accuracy, and recovery. The precisions for the determinations of MDA and OFL, measured in terms of relative standard deviations, were 6.5% and 4.6%, respectively, for intra-day assays and 7.5% and 8.7%, respectively, for inter-day assays. The average recoveries of MDA and OFL spiked in plasma were each close to 100%. The use of this on-line MD-HPLC system permitted continuous monitoring of MDA and OFL in OFL-treated whole blood subjected to UV-A irradiation. Based on our results, the UV-A irradiation markedly increased the level of MDA in the OFL-treated whole blood.     

Automated sampling system for the analysis of amino acids using microfluidic capillary electrophoresis

An improved automated continuous sample introduction system for microfluidic capillary electrophoresis (CE) is described. A sample plate was designed into gear-shaped and was fixed onto the shaft of a step motor. Twenty slotted reservoirs for containing samples and working electrolytes were fabricated on the “gear tooth” of the plate. A single 7.5-cm long Teflon AF-coated silica capillary serves as separation channel, sampling probe, as well as liquid-core waveguide (LCW) for light transmission. Platinum layer deposited on the capillary tip serves as the electrode. Automated continuous sample introduction was achieved by scanning the capillary tip through the slots of reservoirs. The sample was introduced into capillary and separated immediately in the capillary with only about 2-nL gross sample consumption. The laser-induced fluorescence (LIF) method with LCW technique was used for detecting fluorescein isothiocyanate (FITC)-labeled amino acids. With electric-field strength of 320 V/cm for injection and separation, and 1.0-s sample injection time, a mixture of FITC-labeled arginine and leucine was separated with a throughput of 60/h and a carryover of 2.7%.     

Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids

The principle of sequential injection (SI) was exploited to develop a fully automated pre-column derivatization procedure combined on-line to liquid chromatography (LC). Using SI-LC derivatization 14 amino acids were determined fluorimetrically in pharmaceuticals with o-phthaldialdehyde (OPA) as the derivatization reagent. The SI system was used for the handling of samples and reagents, on-line mixing and introduction to the LC injection system. Chemical (pH and reagents concentrations) and instrumental variables (sample and reagent volumes, reaction time and flow rate) were optimized to attain the highest reaction yield and detector signal. Reversed phase chromatographic resolution of 14 amino acids was achieved within 35 min using gradient elution. The automated operation of the coupled SI-LC system resulted in very satisfactory performance. The method was applied for the simultaneous determination of amino acids in pharmaceutical formulations.     

Direct determination of enantiomeric purity of FMOC amino acids with high performance liquid chromatography

Summary Analytical methods are described which allow a direct determination of enantiomeric purity of seventeen FMOC amino acids commonly used in peptide synthesis. The corresponding ester derivatives can be separated directly on cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). The methods are suitable for primary as well as secondary FMOC amino acids. The presence of a highly sensitive fluorescence moiety within the molecule, in combination with large separation factors (-values between 1.5–2.2) allowed for a general detection limit below 0.05%. In several cases the antipode has been determined in the ppm-range. An interesting result has been observed with respect to the elution order of the FMOC amino acid esters. The elution order of the Trp enantiomers is opposite to that obtained with the other amino acids. This is contrary to the generally held belief that elution order is identical within a homologous series of racemates when chromatographed under identical conditions on the same chiral stationary phase. In addition, the inversion of elution of the Pro enantiomers depending on the estertype indicates a competition of different separation mechanisms.     

Reaction of o-phthalaldehyde with amino acids and glutathione. Application to high-performance liquid chromatography determination

Experimental conditions were found for the preparation of stable fluorescent adducts of o-phthalaldehyde with glutathione and its metabolites: glutamine, glutamic and aspartic acids, gamma-glutamylglutamine and gamma-glutamylglutamylglutamine. The structure of the glutathione isoindole derivative obtained was confirmed by NMR studies. The procedure was applied to reversed-phase high-performance liquid chromatographic separation of the previous compounds. The method was extended to glutathione and "total glutathione" determinations.     

Automated solid-phase extraction for determination of amodiaquine, chloroquine and metabolites in capillary blood on sampling paper by liquid chromatography

Summary A bioanalytical method using solid-phase extraction and high-performance liquid chromatography (LC) has been developed and validated for the determination of amodiaquine, chloroquine and their metabolites in 100 μL blood applied to sampling paper. The intra-assay precision for all analytes was <5% at 2000 nM, <7% at 750 nM and <10% at 100 nM. The interassay precision for all analytes was <4% at 2000 nM, <7% at 750 nM and <12% at 100 nM. The lower limit of quantitation was 100 nM for all analytes. The limit of detection was 30 nM in 100 μL venous blood applied to sampling paper.