Derivatization High-performance Liquid Chromatography Research Articles

Quantitation of tryptophan and other plasma amino acids by automated pre-column o-phthaldialdehyde derivatization high-performance liquid chromatography: improved sample preparation

Pre-column derivatization with o-phthaldialdehyde is a rapid and sensitive method for the quantitation of amino acids in biological fluids. This method uses acetonitrile as a deproteinizing reagent which gives improved recovery of tryptophan compared with 5-sulfosalicylic acid and permits the measurement of aspartic acid which coelutes with 5-sulfosalicylic acid. The method is automated to increase reproducibility and convenience. Mean coefficients of variation for peak areas relative to internal standard were 3.2 and 5.2% for amino acid standards and plasma samples, respectively. The presence of nitrilotriacetic acid stabilized the o-phthaldialdehyde reagent which is important in an automated system. The method is suitable for the analysis of large numbers of plasma samples where total tryptophan and aspartic acid are of interest.

Pre-columm fluorescence derivatization high-performance liquid chromatography of opioid peptides in rat brain and its use for enzymatic peptide characterization.

A high-performance liquid chromatographic method involving fluorescence derivatization followed by separation on a reversed-phase polymer (octadecylated polyvinylalcohol copolymer gel) column is described for the determination of opioid peptides in rat brain tissues. The peptides extracted from brain tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate. The derivatives were separated on an Asahipak ODP-50 column by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 9.5). The detection limits (S/N = 3) for the peptides were 0.33-1.21 pmol per 100 microliters injected. The method actually permit the determination of leucine enkephalin, methionine enkephalin, methionine enkephalin-Arg-Phe and methionine enkephalin-Arg-Gly-Leu in the tissues. The method is also applied to the characterization of the peptides in the tissues by means of enzymatic degradations with carboxypeptidase A and trypsin.

High Performance Liquid Chromatographic Determination of Acidic Catecholamine Metabolites in Human Urine Using Postcolumn Fluorescence Derivatization with dl-1,2-Diphenylethylenediamine

dl-1,2-Diphenylethylenediamine was found to react sensitively in an alkaline solution with 3,4-dihydroxymandelic acid as well as other catechol compounds and 4-hydroxy-3-methoxyphenyl compounds to yield fluorescent products. By using this reaction, a postcolumn fluorescence derivatization high performance liquid chromatography of the acidic metabolites of catecholamines (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) in human urine was established. These compounds were separated on an anion-exchanger column (TSK-gel DEAE-2SW) and subjected to derivatization involving coulometric oxidation followed by a fluorescence reaction with the named reagent. The detection limits (S/N=3) for the acidic metabolites in urine were 15–30 fmol in a 100-μl injection volume.

Amino acid analysis using 1-naphthylisocyanate as a precolumn high performance liquid chromatography derivatization reagent

A method which uses 1-naphthylisocyanate as an HPLC precolumn derivatization reagent for amino acid analysis is described. Derivatization is carried out by adding the isocyanate dissolved in dry acetone to a buffered amino acid solution followed by extraction of the excess reagent with cyclohexane. The resulting naphthylcarbamoyl amino acids are stable and highly fluorescent, with excitation maxima at 238 and 305 nm and an emission maximum at 385 nm, for most amino acids. Ultraviolet detection near 222 nm, the absorption maximum, can also be employed. HPLC procedures permitting the analysis of protein hydrolysates, brain extract, cerebrospinal fluid, and blood plasma are presented. The method is particularly suitable for autosampler procedures since samples can be derivatized and diluted in advance and stored at room temperature in the sampler while awaiting injection. Other advantages include high sensitivity, the possibility of recovering the derivatives from the column effluent, and the absence of a reagent peak in the chromatograms.

The effects of two anticonvulsants on amino acid levels in the developing rat cerebellum

Two anticonvulsants were administered pre- and postnatally to determine their effects on putative amino acid neurotransmitter levels in the rat cerebellum. The amino acids were quantitated using precolumn fluorescence derivatization and reverse-phase high performance liquid chromatography at various postnatal intervals. Treatment with clonazepam produced an initial depression in levels of most of the amino acids analyised. By three weeks postnatal all the amino acids, with the exception of GABA, had returned to control levels. GABA levels were still depressed five weeks after the cessation of treatment. Phenobarbital treatment produced an initial elevation in the level of GABA. At three weeks postnatal, both GABA and glutamate levels were elevated and remained so at eight weeks postnatal. In conclusion, the data demonstrated that each anticonvulsant produced unique, acute and chronic alterations in the levels of the cerebellar amino acids.

Ultramicro analysis of bioactive substances: an approach based on the development of highly selective fluorogenic reagents

Highly sensitive methods established in our laboratories are outlined for the determination of bioactive substances including oligopeptides and related enzyme activities (i.e. aromatic aldehydes, their precursors and related amine-metabolizing enzymes, α-keto acids, sialic acids, catecholamines, their metabolites and biosynthetic enzymes, biogenic guanidines, arginine- and tyrosine-containing peptides, reducing sugars, fatty acids involving prostaglandines, nucleosides and nucleic acid bases with active imino hydrogen and biogenic alcoholic compounds) in biological materials, by means of conventional fluorometry and mostly pre- and postcolumn fluorescence derivatization high performance liquid chromatography. A series of mono- and bifunctional fluorogenic reagents has been developed for the selective derivatization of such bioactive substances and as enzyme substrates. Their use in the methods is briefly referred in the text.

Comparison of automated pre-column and post-column analysis of amino acid oligomers

It has been shown that various amino acids will polymerize under plausible prebiotic conditions on mineral surfaces, such as clays and soluble salts, to form varying amounts of oligomers ( n = 2–6). The investigation of these surface reactions required a quantitative method for the separation and detection of these amino acid oligomers at the picomole level in the presence of nanomole levels of the parent amino acid. In initial high-performance liquid chromatography (HPLC) studies using a classical postcolumn o-phthalaldehyde (OPA) derivatization ion-exchange HPLC procedure with fluorescence detection, problems encountered included lengthy analysis time, inadequate separation and large relative differences in sensitivity for the separated species, expressed as a variable fluorescent yield, which contributed to poor quantitation. We have compared a simple, automated, pre-column OPA derivatization and reversed-phase HPLC method with the classical post-column OPA derivatization and ion-exchange HPLC procedure. A comparison of UV and fluorescent detection of the amino acid oligomers is also presented. The conclusion reached is that pre-column OPA derivatization, reversed-phase HPLC and UV detection produces enhanced separation, improved sensitivity and faster analysis than post-column OPA derivatization, ion-exchange HPLC and fluorescence detection.

In vitro release and electrophysiological effects in situ of homocysteic acid, an endogenous N-methyl-(D)-aspartic acid agonist, in the mammalian striatum

A potassium-induced, calcium-dependent release of endogenous homocysteic acid (HCA) from rat striatal slices was demonstrated. A precolumn derivatization high-performance liquid chromatography method was developed that allowed quantitative determination of sulfur-containing amino acids at the picomole level. Intracellular recordings from cat caudate neurons during simultaneous microiontophoretic application of drugs and electrical stimulation of the corticocaudate pathway showed that (L)-HCA evoked a depolarization pattern similar to that induced by N-methyl-(D)-aspartic acid (NMDA), and both these depolarizations could be selectively inhibited by a specific NMDA antagonist, (D)-2-amino-7-phosphonoheptanoic acid [(D)-AP-7]. A selective antagonism of (L)-HCA-induced depolarizations by (D)-AP-7 was confirmed in quantitative experiments with the frog hemisected spinal cord in vitro. Small quantities of iontophoretically applied (L)-HCA, but not of quisqualate, potentiated cortically evoked EPSPs in cat caudate neurons. These observations suggest that (L)-HCA might be a candidate as an NMDA-receptor-preferring endogenous transmitter in the caudate nucleus. One possible function for such transmitter systems could be the enhancement of EPSPs.

Fluorescence detection of 8-hydroxyquinoline and some of its halogenated derivatives using post-column derivatization in high-performance liquid chromatography

Fluorescence detection of 8-hydroxyquinoline and some of its halogenated derivatives using post-column derivatization in high-performance liquid chromatography

Fluorometric detection and determination of carbohydrates by high-performance liquid chromatography using ethanolamine

Reaction of a mixture of ethanolamine and boric acid with reducing sugars gave intense fluorescence. Application of this reaction to the postcolumn derivatization in high-performance liquid chromatography provided a rapid, sensitive, and simple detection and determination of carbohydrates under a mild reaction condition. Separation and determination of carbohydrates in enzymatic hydrolysate of amylose and human serum are also described.

Automation of pre-column derivatization in high-performance liquid chromatography : Application to ion-pair partition chromatography

An automated pre-column derivatization system is described. Problems arising from the coupling of this AutoAnalyzer-type system with high-performance liquid chromatography are discussed in detail. The system has been applied to ion- pair partition chromatography. The ion-pairs of a mixture of some alkaloids (apoatropine, hyoscyamine, scopolamine and ergotamine) were formed with picric acid in an aqueous phase, extracted into chloroform and injected on-line on to a silica gel column for separation within 15 minutes. This study demonstrates the possibility of obtaining an automated pre-column derivatization system with a good relative standard deviation of <3% per injection ( n = 6). The linearity of the system for hyoscyamine and ergotamine is significant in the concentration ranges of 80–200 ng and 240–720 ng per injection, respectively. The experience gained is of general validity and could generated more studies in this area.

Post-column derivatization in high-performance liquid chromatography using the air segmentation principle: application to digitalis glycosides

The possibility of using post-column derivatization techniques to improve the detection in high-performance liquid chromatography (HPLC) has been suggested before, but a serious limitation to the choice of the reactions is the kinetics, as slow reactions require longer reaction times and correspondingly larger reaction units (long spirals) with the associated problems such as a high pressure drop and loss of resolution. In this study it was attempted to overcome some of these drawbacks by applying the well known air segmentation principle used in connection with Auto-Analyzers. The studies were carried out on cardiac glycosides which, upon reaction with concentrated hydrochloric acid, yield highly fluorescent products. As this reaction is kinetically slow, with at least a 10-min reaction time for a reasonable fluorescence yield, it served ideally for the investigation. The optimal reaction conditions and the influence of solvents, reagent composition, temperature and time on the kinetics and fluorescence yield were studied. With a 10-min reaction time the band broadening was only about 15%. The reproducibility of the derivatization step is 1.2% (relative standard deviation, n = 10) and the improvement in the detection limit was at least 100-fold in comparison with UV detection. For a non-retained glycoside the detection limit is 500 pg per injection at a signal to noise ratio of 4:1. The technique was applied successfully to members of the C-group of cardiac glycosides (digoxin, digoxigenin, lanatoside C and desacetyl-lanatoside C), which were separated on a reversed phase HPLC system. Many of the concepts studied are of general validity for post-column reactions with relatively slow kinetics.

New aspects of post-column derivatization in high-performance liquid chromatography

A nonapeptide-Fluram reaction system has been further investigated in order to gain a general insight into the problems of post-column derivatization. Some kinetic aspects and their influence on reaction volumes, design of reaction units and mixing devices have been studied. As a result, reaction times up to several minutes can be tolerated with the present system without appreciable band broadening. The coupling of sample enrichment phenomena with post-column reactions for further improvement of detection properties was also studied. Several millilitres of sample solution can be injected without serious band broadening. The combination of step gradients with post-column reactions and pre-concentration techniques has been shown to be feasible and opens up interesting possibilities for trace analysis in complex matrices. The reproducibility of retention times was less than 1.5% relative standard deviation and of peak areas less than 4% relative standard deviation for all of these operations, thus permitting the characterization and quantitation of substances.

Post-column fluorescence derivatization of peptides : Problems and potential in high-performance liquid chromatography

Some critical parameters such as the pumping system, mixing devices and detector design in instrumentation for post-column derivatization in high-performance liquid chromatography are discussed. The derivatization was studied with pharmaceutically important nona-peptides containing primary amino groups which react with Fluram ® and reaction parameters such as pH, solvent and reagent concentration were investigated. Both adsorption systems and reversed-phase systems were used to separate the peptides prior to the post-column reaction. Reversed-phase chromatography has the advantage of simpler sample preparation, better reaction control and optimization of solvent conditions. As a result, detection limits of between 5 and 10 ng per injection can be obtained and the reproducibility of the results is better than ±2% (relative standard deviation). The method has been applied to the analysis of injection solutions (ampoules).