Three-dimensional HPLC System Research Articles

Enantioselective Analysis of Lactate, Hydroxybutyrates and Malate in Human Physiological Fluids Using a Three-Dimensional HPLC System

Enantioselective Analysis of Lactate, Hydroxybutyrates and Malate in Human Physiological Fluids Using a Three-Dimensional HPLC System

Development of an enantioselective three-dimensional HPLC system for the determination of alanine, valine, isoleucine, allo-isoleucine and leucine in human plasma and urine

A three-dimensional (3D) HPLC system was designed/developed for the discriminative determination of aliphatic chiral amino acids, namely, alanine (Ala), valine (Val), isoleucine (Ile), allo-Ile (aIle) and leucine (Leu), in human physiological fluids. These aliphatic amino acid enantiomers are expected to be new physiologically active molecules and/or biomarkers in mammals. Among these aliphatic amino acids, the structural chain isomers of Leu (Ile/aIle/Leu) have similar chemical-physical properties, and the analytical method for these aliphatic amino acids is required to be highly enantio- and chemo-selective. In the present study, a reversed-phase column (Singularity RP18, first dimension) and a mixed-mode column (Singularity MX-103, second dimension) were utilized to separate these aliphatic amino acids as their scalemic mixtures, then chiral separations were performed using a Pirkle-type enantioselective column (Singularity CSP-001S) in the third dimension. By using the 3D-HPLC system, these aliphatic amino acid enantiomers were completely discriminated, and the analysis of these aliphatic chiral amino acids in the human plasma and urine was successfully carried out. The obtained amounts of the aliphatic amino acids (and %D value, the percentage of D-form to D + L forms) in the human plasma were 2.1 µM (0.5%) for D-Ala, and trace/not-detected for the other D-forms. In human urine, the values were 102.0 µM (24.7%) for D-Ala, 2.0 µM (3.4%) for D-Val, 2.3 µM (10.8%) for D-aIle (%D value of D-aIle was calculated using D-aIle and L-Ile) and 3.3 µM (5.5%) for D-Leu. The present 3D-HPLC system is a powerful and well validated tool for the simultaneous determination of aliphatic amino acid enantiomers, and further biological and clinical studies are expected.

Development of a three-dimensional HPLC system for acidic amino acid enantiomers and determination of their amounts in mice lacking D-aspartic acid oxidase activity

A highly-selective three-dimensional (3D) HPLC system has been designed/developed for the determination of acidic amino acid enantiomers, i.e., aspartic acid (Asp) and glutamic acid (Glu). The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 ×250 mm) in the first dimension, a mixed-mode column (Singularity MX-001, 1.5 ×250 mm) in the second dimension and a Pirkle-type enantioselective column (Singularity CSP-011S, 1.5 ×250 mm) in the third dimension. By using this system, the amounts of Asp and Glu enantiomers in 6 tissues (cerebrum, heart, lung, liver, kidney and testis) and 2 physiological fluids (plasma and urine) were determined in the control C57BL/6J mice and B6DDO-/- mice lacking D-aspartic acid oxidase activity. In the control C57BL/6J mice, a high amount of D-Asp (466.15 nmol/g) was observed in the testis, and relatively high amounts of D-Asp were observed in the cerebrum, lung, liver and kidney (21.40–92.87 nmol/g). In the heart and 2 physiological fluids, the amounts of D-Asp were low (trace–7.75 nmol/g or mL). In the B6DDO-/- mice, the amounts of D-Asp drastically increased in all the tissues and physiological fluids. In contrast to the results of D-Asp, the amounts of D-Glu did not change depending on the alteration of the DDO activity, and the amounts were extremely low and not detectable in most of the tested tissues and physiological fluids. Although the amounts of D-Glu were low, it is noteworthy that clear localization in the testis (8.60–9.53 nmol/g) was demonstrated in both the C57BL/6J and B6DDO-/- mice.

Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV

d-Amino acids (d-AAs) have been associated with age-associated conditions in the general population but their relevance in people with HIV (PWH), who experience accentuated/accelerated aging has not been studied. We compared d-AA levels in HIV-infected and uninfected controls and explored their association with markers of immune activation, gut permeability and organ dysfunction. Case-control analysis. Plasma samples from 60 antiretroviral therapy-treated HIV-infected individuals and 59 uninfected controls were analysed. A three-dimensional HPLC system was used to measure d-and l-asparagine, serine, alanine and proline and presented as %d-AA. Additionally, cell-associated and soluble markers of immune activation and senescence were characterized. Kidney and liver functions were expressed as estimated glomerular filtration rate and fibrosis-4 scores, respectively. Mann-Whitney and Spearman rank correlation coefficients were used for statistical analysis. d-Asparagine, d-serine, d-alanine and d-proline were detectable in all plasma samples and correlated with age in HIV-infected and uninfected but not different between groups. Kynurenine/tryptophan ratio was positively correlated with all %d-AAs in PWH and with %d-serine and %d-proline in controls. %d-AAs were not consistently correlated with markers of gut permeability in both groups. All %d-AAs were also correlated with kidney function in both groups whereas age-associated accumulation of %d-asparagine, %d-serine and %d-proline were correlated with liver function and the VACS score in controls. Plasma d-AAs are associated with chronological age and correlated with markers of immune activation and organ decline, though variably, in PWH and controls. Their role in the biology of aging warrants further investigation.

Development of a selective three-dimensional HPLC system for enantiomer discriminated analysis of lactate and 3-hydroxybutyrate in human plasma and urine

For the enantiomer discriminated determination of lactate (LA) and 3-hydroxybutyrate (3HB) in various complicated samples, a three-dimensional HPLC (3D-HPLC) system has been designed and developed by investigating the separation of the target analytes from unknown substances observed in the real target matrices. LA and 3HB were pre-column derivatized with 4-nitro-7-piperazino-2,1,3-benzoxadiazole for the sensitive fluorescence detection and introduced into the 3D-HPLC system composed of reversed-phase, mixed-mode and enantioselective separations. The present method was validated by calibration curves, precision and accuracy using standard solutions and human samples, and sufficient values were obtained. Using the method, the levels of d-LA, l-LA, d-3Hb and l-3HB were determined, and their concentrations were 9.9, 1004.2, 79.7 and 2.1 μM in the human plasma and 16.0, 86.6, 8.7 and 4.8 μM in the human urine, respectively. The present 3D-HPLC system could selectively determine trace amounts of the target hydroxy acid enantiomers without disturbance of the intrinsic interfering substances in complicated matrices and the applications to various disease samples are expected.

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

Targeted three-dimensional liquid chromatography: A versatile tool for quantitative trace analysis in complex matrices

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as ‘coupled-column’ or ‘heartcutting’, has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC–MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.

Melatonin in the Testis of the Cabbage Armyworm, Mamestra brassicae

N-Acetyl-5-methoxytryptamine (melatonin; MEL) was detected in the testis of non-diapausing pupae and in the testis of post-diapause pharate adults of the cabbage armyworm, Mamestra brassicae, by use of a threedimensional HPLC system with multiple coulometric electrochemical detectors. Intermediates providing evidence of various metabolic pathways were identified, as follows: tryptophan (TRP) → 5-hydroxytryptophan (5-HTP) → 5-hydroxytryptamine (5-HT) → N-methyl-5-hydroxytryptamine (N-MET) ↳5-hydroxyindoleacetic acid (5-HIAA) ↳N-acetyl-5-hydroxytryptamine (N-ACET-5-HT) ↳melatonin (MEL). The possible physiological roles of melatonin in the testis of both diapausing and non-diapausing pupae are discussed.

Biogenic Monoamines in the Brain and the Corpus Cardiacum Between Albino and Normal Strains of the Migratory Locust, Locusta migratoria

Biogenic monoamines and related compounds were analyzed in the brain and the corpus cardiacum of normal and albino strains of the migratory locust, Locusta migratoria, using a three-dimensional HPLC system with multiple coulometric electrochemical detection. Evidence for the following metabolic pathways was obtained: tyrosine-4 → dihydroxyphenylalanine ( l-dopa) → dopamine → norepinephrine → epinephrine; dopamine → 3-0-methyl dopa → homovanillic acid, and dopamine → 3,4-dihydroxyphenylacetic acid → homovanillic acid in the case of catecholamines; tyrosine-4 → tyramine → hydroxyphenylacetic acid and tyramine → octopamine in the case of phenolamines; and tryptophan → 5-hydroxytryptamine → 5-hydroxyindoleacetic acid and tryptophan → kynurenine in the case of indolealkylamines. The proposed kynurenine pathway is reported for the first time in the neuroendocrine system of an insect. The clearest difference between the albino and control insects was associated with kynurenine. Kynurenine was absent from brain and corpus cardiacum of the fifth instar nymphs on day 0. It was found on day 1 of the fifth nymphal instar and on day 0 of adulthood. However, in albino nymphs, the level was half of that in the controls and kynurenine was not detected in the female brain on day 1. The possible involvement of kynurenine in the development of body color is discussed.

Levels of Biogenic Amines in the Brain during Pupal and Adult Development of the Silkworm, Bombyx mori

Abstract Levels of a wide range of biogenic amines and related metabolites were determined in the brain of the silk­ worm, Bomby mori, during pupal and adult development using a three-dimensional HPLC system with multiple coulometric electrochemical detection. In the brain of the female adults, metabolic pathways such as tyrosine (TYR-4)->dihydroxyphenylalanine (L -DOPA)-dopamine (DA), TYR-4->tyramine (TYRA), and tryptophan (TRP)->5-hydroxytryptamine (5-HT) were identified. At this stage, 3,4-dihydroxyphenyleth-ylene (DOPAC) was also detected. Metabolic pathways of biogenic amines in the brain from pupal to adult stages are discussed.

Metabolism of biogenic monoamines in the ciliated protozoan, Tetrahymena pyriformis.

1. The three-dimensional HPLC system was used to detect the presence and determine the levels of biogenic monoamines, including precursor amino acids and metabolites, simultaneously in an extract of the ciliated protozoan, Tetrahymena pyriformis. 2. Representative biogenic monoamines, such as dopamine (DA), 5-hydroxytryptamine (5-HT), epinephrine (E) and kynurenine (KYN) were found to be synthesized in Tetrahymena and released into the growth medium. 3. The following metabolic pathways were suggested to be operative: (L-DOPA)-DA-(Epinine)-E-dihydroxyphenylethyleneglycol (DOPEG)-vanillylmandelic acid (VMA) and tyrosine-4 (TYR-4)-tyramine (TYRA)-hydroxyphenylacetic acid-4 (HPAC-4) in the case of catecholamines, and tryptophan (TRP)-5-HT and TRP-KYN-xanthurenic acid (XA) in the case of indolalkylamines. 4. As judged from the released metabolites, systems for generation of biogenic monoamines in Tetrahymena seem to be active during the logarithmic phase of its growth.

Simultaneous analyses of monoamines and their metabolites in urine specimens of patients with neuroblastoma

1. Tyrosine and tryptophan, as well as 26 metabolites of these amino acids, were analyzed simultaneously in urine specimens from patients with neuroblastoma and control infants by a three-dimensional HPLC system to develop an early diagnosis. 2. Levels of detected compounds in urine from patients with neuroblastoma were generally higher in the case of catecholamines and lower in the case of indolalkylamines than those in controls. 3. The pathways of Dopamine-3,4-Dihydroxyphenylacetic acid-Vanillylmandelic acid, Dopamine-3-Methoxy-4-hydroxyphenylethylene glycol-Vanillylmandelic acid-Vanillic acid and Tyrosine-4-Hydroxy-phenylacetic acid-4 were, in particular, found to be active in patients with neuroblastoma.

Biogenic amine levels in the central nervous system and haemolymph of the silkworm, Bombyx mori

1. A wide range of biogenic amines and related metabolites were quantified in the silkworm, Bombyx mon from fourth instar to post-spinning stage using a three-dimensional HPLC system with multiple coulometric electrochemical detection. 2. In the central nervous system, metabolic pathways such as tyrosin (TYR-4)-dopa ( L-DOPA)-dopamine (DA), TYR-4-tyramine (TYRA), tryptophan (TRP)-5-hydroxytryptamine (5-HT) were found. In some stages, normetanephrine (NMN), 3,4-dihydroxyphenylethylene (DOPAC) and 5-hydroxyindoleacetic acid (5HIAA) were detected. 3. In the haemolymph, TRY-4-L-DOPA-DA pathway was mainly found. 3-Methoxy-4-hydroxy- phenyl glycol (MHPG) in fourth instar and norepinephrine (NE) in fifth instar were also detected. 5-HT was lacking. 4. Metabolic pathways of biogenic amines in the central nervous system and haemolymph were discussed.