Stanley Lam

Reversed-phase high-performance liquid chromatographic resolution of d- and d-Dns-amino acids by mixed chelate complexation.

Abstract Separation of optical isomers of Dns derivatives of amino acids by reversed-phase high-performance liquid chromatography has been accomplished by adding a complex of an optically active amino acid to Cu(II) to the mobile phase. Cu(II) complexes of l -proline, l -arginine and l -histidine in the mobile phase yielded different degrees of separation. The concentrations of acetonitrile and the Cu(II) ligand as well as the pH all affect the separations. A chromatographic model is proposed that is based on the formation of ternary complexes by the d -Dns-amino acids and the chiral additive associated with the stationary phase. The separation selectivity appears to be based on different steric interactions between the alkyl side chains of the amino acids and the chiral additive.

QT prolongation and the antiarrhythmic efficacy of amiodarone

Amiodarone is an antiarrhythmic agent known to cause prolongation of action potential duration which is reflected in the electrocardiogram as a prolongation of the QT interval. Prolongation of the QT interval in patients dying suddenly was compared with that in patients who remained alive to determine whether a difference existed between these two groups. The electrocardiogram and amiodarone levels were evaluated in 33 patients who presented with cardiac arrest and symptomatic ventricular tachycardia in whom no other antiarrhythmic agent was found effective in preventing induction of ventricular tachycardia during electrophysiologic studies. There were 30 men and 3 women (mean age 52 +/- 10 years). Twenty-three are alive after a mean follow-up period of 12 +/- 7 months. Ten died: six suddenly, three of non-cardiac causes and one of congestive heart failure. Using a two-way analysis of variance, the percent change in QT, QTc, JT and JTc intervals before and after amiodarone therapy was analyzed. Marked prolongation in the QT interval was present in patients who remained alive with amiodarone therapy. A significant difference in percent QT prolongation was seen between the latter patients and those who died suddenly (p less than 0.005). No difference was observed in the percent change in QRS interval between the two groups. The levels of amiodarone (2.5 versus 3.2 micrograms/ml) and its metabolite (desethylamiodarone) were not significantly different between the living patients and those who died suddenly. These findings suggest that a prolongation of the QT interval may be a marker for the therapeutic antiarrhythmic effect of amiodarone.

Stereoselective D- and L-amino acid analysis by high-performance liquid chromatography

This paper describes a gradient system for separating D- and L-isomers of Dns-amino acids by mixed chelate complexation through the addition of histidine methyl ester and copper sulfate to the mobile phase. Most of the biologically important amino acids were separated in a single analysis. With a simple solvent gradient consisting of increasing concentrations of acetonitrile in L-histidine methyl ester buffer all the common amino acids were resolved except cysteine and all optical isomers were resolved except those of threonine, alanine and proline. Analysis time was 90 min. Use of this system for determining non-protein amino acids in human cerebrospinal fluid showed the amino acids to be L-isomers, as expected. The pattern in fluid from a patient with bacterial meningitis was different from that of most of the others.

Resolution of optical isomers of Dns-amino acids by high-performance liquid chromatography with l-histidine and its derivatives in the mobile phase

Abstract This paper describes our continuing work in resolving the optical isomers of Dns-amino acids by high-performance liquid chromatography with mixed chelate complexation. Addition of copper(II) complexes of l -histidine to the mobile phase resulted in resolution of many d - and l -Dns-amino acids, including those with aliphatic, polar and aromatic substituents. With polar substituents, highly selective incorporation of the l -enantiomer into the ternary complex with increased retention on the column was observed. The reverse occurred with amino acids with aliphatic side chains; the d -isomers were incorporated preferentially. With aromatic substituted amino acids, the resolution was pH dependent. Substitution of copper(II) complexes of l -histidine methyl ester in the mobile phase dramatically reduced the stereoselectivity, although the isomers were still resolved. Copper(II) complexes of N-acetyl- l -histidine used in the mobile phase gave no stereoselectivity. Excellent separations of isomers were achieved with several of these systems. Many pairs of amino acids could be separated in the same chromatographic analysis.

High-performance liquid chromatography of amino acids in urine and cerebrospinal fluid.

Two different methods for analyzing amino acids by reversed-phase high-performance liquid chromatography (HPLC), both of which can separate D- and L- stereoisomers, have been used for studying the amino acid composition of cerebrospinal fluid (CSF) and urine. One method, by which Dns derivatives of amino acids are separated as mixed chelate complexes with Cu(II) and a single stereoisomer of a second amino acid, was used to analyze CSF. CSF contains ca. 10 mumole/l per amino acid, compared to 100 mumole/l in serum. The high sensitivity of fluorescence detection enabled complete analysis, starting with 50 microliter of fluid. The second method, which uses lower concentrations of both the copper and the second amino acid and detects amino acids by the change in absorbance of the copper complex, was used to measure the urine concentration of the lysine metabolite, pipecolic acid (piperidine-2-carboxylic acid), a secondary amino acid that is difficult to detect by the more usual detection methods. Our procedure involves passing urine through a cation-exchange column, collecting the fraction containing pipecolic acid, and chromatographing it on a reversed-phase HPLC column with a mobile phase containing L-aspartame and Cu(II). To assess the utility of the method, urine samples from a patient given loading doses of D- or L-isomers were analyzed. When either isomer was administered, both D- and L-isomers were detected, but in different proportions. Varying proportions and concentrations of both isomers were also detected in the urines of patients with hyperpipecolatemia from different metabolic abnormalities.

Resolution of D- and L-amino acids after precolumn derivatization with o-phthalaldehyde by mixed chelation with Cu(II)-L-proline☆

Abstract Highly selective separation of amino acids can be accomplished by reversed phase chromatography of mixed chelate complexes of the analyte acids with equimolar concentrations of Cu(II) and an optically active second amino acid in the mobile phase. Resolution of D - and L -Dns-amino acids was accomplished this way using L -proline or L -histidine as the second amino acids. We have now extended the mixed chelation approach to resolve o-phthalaldehyde (OPA) derivatives. The free amino acid was reacted with OPA in the presence of N-acetyl- L -cysteine. High-performance liquid chromatography of the derivative was then perfortned on a reversed-phase column, with a mobile phase containing L -proline and Cu(II), using fluorescence detection. All primary amino acids reacted rapidly with OPA without measurable side products. The optical isomers were resolved. Study of the mechanism of optical selectivity confirmed that the chiral sulfhydryl reagent was responsible for the formation of diastereomeric mixed chelate complex and for the resolution of the isomers. The same approach was also applied to resolve stereoisomers of amines and sulfhydryl compounds.

Resolution of D,L-Dansyl Amino Acids by HELC with a Cu(II)-L-proline Eluant

Abstract Highly fluorescent dansyl derivatives of D and L amino acids were resolved on a reversed phase column by using an aqueous eluant containing copper(II)-L-proline.

Resolution of Optical Isomers as the Mixed Chelate Copper(III) Complexes by Reversed Phase Chromatography

Abstract Highly selective separation of D and L amino acids can be effected by reversed phase chromatography of mixed chelate complexes of the analyte acids with equimolar concentrations of Cu(II) and an optically active second amino acid in the mobile phase. The stabilities of the formed diastereomeric ternary complexes will determine the resolution of enantiomers. By this approach, amino acids were resolved as the dansyl and O-phthalaldehyde (OPA) derivatives and imino acids were separated without derivatization. Resolution of D and L dansyl amino acids was accomplished as the mixed complexes of Cu(II) with L-proline, L-arginine, L-histidine and L-histidine methyl ester as the second amino acids. Among the chiral ligands we studied, L-histidine methyl ester is unique in that it possesses both achiral selectivity for the dansyl amino acids and chiral selectivity for the respective D and L enantiomers. With a mobile phase gradient of acetonitrile in a buffer containing Cu(II) L-histidine methyl ester comp...

L-pipecolaturia in Zellweger syndrome

Purified rat peroxisomes have been reported to oxidize D-pipecolic acid and the pipecolaturia of Zellweger syndrome has been attributed to the absence of peroxisomes. The logical consequences would be excesses of D-pipecolic acid in the urine of patients with Zellweger syndrome. The urine of two patients with Zellweger syndrome has been analyzed by complexing the pipecolic acid to copper-aspartame to separate the L- and D-isomers. L-Pipecolic acid constituted 100% and 78% of the total pipecolic acid in the two urines. The possibility of preferential retention of D-pipecolic acid was excluded by measuring renal excretion in two control subjects following administration of each isomer. The clearance of L-pipecolic acid was 1.1 and 0.2 ml/min and of D-pipecolic acid was 36.4 and 43.6 ml/min. These results do not support the contention that the pipecolaturia of Zellweger syndrome is the direct result of peroxisomal deficiencies.

High-performance liquid chromatography of hydroxy-steroids detected with post-column immobilized enzyme reactors

Assaying the low concentrations of steroid hormones in extracts of body fluids requires detectors that are both highly sensitive to the steroid and relatively insensitive to interfering compounds usually present in much higher concentrations. To explore the use of moderately specific enzymes in post-column reactors, we immobilized 3 alpha- and beta-hydroxysteroid dehydrogenase on controlled pore glass beads, 37 microns in diameter, and constructed 4.6-mm diameter reactor columns, 3-cm long, packed with one of the two kinds of these beads. Hydroxysteroids eluted from the analytical column were mixed with the coenzyme, nicotinamide adenine dinucleotide (NAD), before passing through the reactor. The effluent from the reactor was passed through the 70-microliters flow cell of a fluorometer in which the fluorescence of the NADH produced in the enzyme-catalyzed oxidation of the hydroxysteroid was monitored. At the conventional high-performance liquid chromatography flow-rates used, oxidation of the steroids was almost complete. The yield depended on both the residence time of steroids in the reactor column and the concentration of organic modifier in the reaction mixture. Maximal yield was obtained with buffer having a low organic solvent concentration and passing through the reactor slowly. In assays of mixtures of epimeric hydroxysteroids, the 3 alpha-hydroxysteroids were detected with the 3 alpha-hydroxysteroid dehydrogenase reactor; the beta-hydroxysteroids were not, confirming the specificity of the enzymatic detection. With the fluorometer used, picomole quantities of steroids could easily be distinguished from noise.