Enhanced Separation Capability of Sequential Injection Chromatography for Fluorimetric Determination of Intracellular Dissolved Free Amino Acids in Marine Microalgae.

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50 × 4.6 mm C18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10 mM phosphate buffer (pH 7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10 mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30 min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.

Improvements in the separation capabilities of sequential injection chromatography: determination of intracellular dissolved free amino acid profiles in three taxonomic groups of microalgae.

INTRODUCTION: Dissolved free amino acids (DFAA) in intracellular extracts of marine microalgae can be determined by sequential injection chromatography (SIC). This technique uses portable, low-cost instrumentation but its applications have been limited to short monolithic columns because of components not resistant to high pressures. OBJECTIVE: To develop a SIC method for determination of DFAA by exploring an instrument modified to handle pressures of 1000 psi. METHOD: The method was based on pre-column derivatisation of the amino acids with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 9.4), separation and fluorimetric detection (λ(excitation)= 340 and λ(emission)= 450 nm). Separation was achieved by stepwise gradient elution using six mobile phases. The first elution step used a mobile phase composed of methanol:tetrahydrofuran:10 mm phosphate buffer (pH 7.2) at a volumetric ratio of 8:1:91. Additional elution steps used mobile phases containing methanol and 10 mM phosphate buffer at ratios of 17.5:82.5, 25:75, 35:65, 50:50 and 65:35. RESULTS: Nineteen chromatographic peaks were observed in a mixture of 20 amino acids. The only complete co-elution was between tryptophan and methionine. Detection limits varied from 0.10 µm for isoleucine to 1.5 µm for lysine. Recoveries from spiked extracts were between 84 and 131%. CONCLUSION: Resolutions of the amino acid pairs glutamine and histidine, valine and phenylalanine, and isoleucine and leucine were 1.5, 0.75 and 1.3, respectively. The proposed method found different profiles of DFAA among the three species of algae, suggesting its adequacy for metabolic studies.

Determination of apramycin in oral soluble powder by a HPLC method using pre-column derivatization with o-phthalaldehyde and UV detection / Determinação da apramicina em pó solúvel oral por um método de HPLC utilizando pré-coluna de derivação com o de detecção de ftalaldeído e UV

A high-performance liquid chromatographic method employing pre-column derivatization with o-phthalaldehyde (OPA) and 2-mercaptoacetic acid was developed for the determination of apramycin, an aminoglycoside antibiotic used in veterinary medicine, in the oral soluble powder form. The chromatographic separation was done by ion-pair HPLC using a C18 reversed-phase column, Synergy Hydro (150 mm x 4.6 mm x 4 µm) and mobile phase composed of 0.005 mol/L sodium octanosulfonate in a mixture of acetonitrile: water: acetic acid (45:55:2) (v/v/v) with a flow rate of 1.0 mL/min; the UV detector was operated at 332 nm. The developed method was validated according to official compendia guidelines, having demonstrated robustness, selectivity and linearity for the concentration range of 0.02 to 0.05 mg/mL, precision (with RSD < 2.0 percent both for intra and inter-day precision) accuracy (average recuperation of 99.33 percent) and detectivity (quantification and detection limits of 0.08 and 0.02 µg/mL, respectively). Three batches of commercial apramycin oral soluble powder were analyzed by both the proposed method and the official microbiological method, where all the results obtained were in the acceptable range (95 percent to 105 percent of labeled value of apramycin). Both methods were statistically compared by the t test, which yielded no significant differences (α = 0.05) thereby confirming the equivalence of the methods.

Foi desenvolvido um método por cromatografia líquida alta eficiência empregando derivatização pré-coluna com o-ftalaldeído (OPA) e ácido mercaptoacético para determinação de apramicina, um antibiótico aminoglicosídeo de uso veterinário, em pó oral solúvel. A separação cromatográfica foi feita por fase reversa com pareamento iônico utilizando-se coluna Synergy Hydro C18 (150 x 4,6 mm x 4 µm) e fase móvel composta por octanossulfonato de sódio 0,005 mol/L em mistura de acetonitrila:água:ácido acético nas proporções 45:55:2 (v/v/v), numa vazão de 1.0 mL/min; efetuou-se detecção por UV a 332 nm. O método foi validado de acordo com os compêndios oficiais e demonstrou robustez, seletividade, linearidade na faixa de 0,02 a 0,05 mg/mL, precisão (com DPR < 2,0 por cento tanto para a precisão intra-dia quanto para a precisão inter-dia), exatidão (recuperação média de 99,33 por cento) e detectabilidade (limite de quantificação e de detecção iguais a 0,08 e 0,02 µg/mL, respectivamente). Analisaram-se 3 lotes de apramicina pó oral solúvel pelo método proposto e pelo método microbiológico oficial e todos os resultados obtidos estavam dentro do limite de aceitação (95 por cento-105 por cento valor rotulado de apramicina). Ambos os métodos foram comparados estatisticamente pelo teste t, não sendo encontradas diferenças significativas entre eles para α=0,05, sendo os dois equivalentes.

Improved high-performance liquid chromatographic method for GABA and glutamate determination in regions of the rodent brain.

A C18 reversed-phase column and isocratic fluorescence HPLC method for the simultaneous detection of glutamate and gamma-aminobutyric acid (GABA) is described. In this article a fast and more efficient method for the extraction of these neurotransmitters in rat brain tissue is also presented. The supernatant was derivatized with o-phthalaldehyde (OPA) and analyzed by HPLC with fluorescence detection. Intraday reproducibility was 97.0% and 96.7% and interday reproducibility was 97.1% and 93.7% for GABA and glutamate, respectively. Recovery assays indicate that the accuracy of the method for GABA is 99.6+/-2.3% and for glutamate is 101.9+/-1.8%. In addition, the time consumed to run a sample is lower than that described by other authors. Mean elution time was 3.10 min and 8.22 min for glutamate and GABA, respectively. Thus, in a total runtime of less than 9 min both neurotransmitters were detected. Moreover, when compared to the current methods, the extraction solution used here allowed a high drawing out of the neurotransmitters, glutamate and GABA, from the hippocampus, thalamus and prefrontal cortex of the rat brain.

A non-radiolabeled heme-GSH interaction test for the screening of antimalarial compounds.

Intraerythrocytic Plasmodium produces large amounts of toxic heme during the digestion of hemoglobin, a parasite specific pathway. Heme is then partially biocristallized into hemozoin and mostly detoxified by reduced glutathione. We proposed an in vitro micro assay to test the ability of drugs to inhibit heme-glutathione dependent degradation. As glutathione and o-phthalaldehyde form a fluorescent adduct, we followed the extinction of the fluorescent signal when heme was added with or without antimalarial compounds. In this assay, 50 microM of amodiaquine, arthemether, chloroquine, methylene blue, mefloquine and quinine inhibited the interaction between glutathione (50 microM) and heme (50 microM), while atovaquone did not. Consequently, this test could detect drugs that can inhibit heme-GSH degradation in a fast, simple and specific way, making it suitable for high throughput screening of potential antimalarials.

Interaction of aluminium ions with some amino acids present in human blood.

The interaction of aluminium with some amino acids present in human blood was studied combining ion-chromatography (IC), atomic absorption spectrometry (AAS) and ultrafiltration (UF) techniques. An IC system for simultaneous determination of ornithine, lysine, glutamic acid, aspartic acid and tyrosine was developed. By adding aluminium to standard solutions of the amino acids and keeping the pH at 6 and 7 it was possible to verify that aluminium caused a reduction on the amino acid chromatographic signals. Similar experiment, carried out for copper showed the same behaviour (with different percentage of signal reductions) and validated the results for aluminium, considering that the interaction Cu-amino acid is well-established. The AAS analysis of sample fractions (500 microl) after the IC separation showed that aluminium (as copper as well) is not present in the fractions in which the amino acid peaks appear in the chromatogram. These approaches carried out with serum samples after UF showed that part of the "free" fraction of serum aluminium is distributed, besides other ligands, among these amino acids. It was found that in serum the affinity for aluminium followed the sequence Lys>Orn>Tyr>Glu approximately Asp.

High-performance liquid chromatographic determination of mexiletine enantiomers in plasma using direct and indirect enantioselective separations.

Two methods were developed for the determination of mexiletine enantiomers in plasma samples suitable for studies on the stereoselective disposition of this drug. Both methods used fluorescence detection to improve sensitivity and selectivity. The direct enantioselective separation was based on the chiral resolution of mexiletine-2-naphthamide derivatives on a Chiralcel OJ column. The calibration curves were linear over the concentration range 50-500 ng/ml for each enantiomer; therefore the method can be used only for therapeutic monitoring, drug interaction and multiple dose pharmacokinetic studies. The indirect method was based on the formation of diastereomers using o-phthaldialdehyde and N-acetyl-L-cysteine reagents. The diastereomers were resolved on a reversed-phase RP-18 column. The method proved to be suitable for single or multiple dose pharmacokinetic studies based on the low quantification limit (1 ng/ml) and the broader linear range (1-1000 ng/ml) obtained.