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A method is described for the simultaneous determination of vanilmandelic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 5-hydroxyindoleacetic acid, and homovanillic acid in a human plasma sample using reversed-phase high-performance liquid chromatography with column switching and amperometric detection. Two methods of sample preparation were tested. Liquid-liquid extraction yields better recoveries, is more selective and precise than solid-phase extraction and allows a shorter time of chromatographic analysis. Estimated plasma values of the metabolites from healthy controls are in good agreement with previously reported levels. Studies of alcoholics at the beginning of the delirium tremens provided different plasma levels of the metabolites, dependent on the different duration--and hence the severity--of the delirium.

The simultaneous measurement of homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in human plasma by an ultrafiltration and microbore high-performance liquid chromatography-electrochemical detection technique is established. Conventional preparation of blood is very tedious and time-consuming, but isocratic separation of the analytes in plasma ultrafiltrates using a microbore column could be achieved within 10 min. Hence, theoretically, over 140 analyses can be performed in a working day. The detection limit (signal-to-noise ratio = 3) of this method is about 0.1-0.5 pg per injection for all analytes. The required volume of plasma samples can be less than 100 microliters. Hence, blood loss is minimal, especially in repeated blood sampling. This rapid, simple and sensitive method can, therefore, be used as a routine clinical tool in the simultaneous measurement of plasma homovanillic acid, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid.

A liquid chromatographic analysis with electrochemical detection of 5-methoxytryptamine, 5-methoxytryptophol, 5-methoxyindoleacetic acid and melatonin is described. Optimal elution conditions were determined by studying several variables: pH, buffer salt, counter ion and organic modifier. Measurement of 5-methoxyindoles in the pineal gland and plasma of hamsters has been performed after extraction. This method is specific and sensitive, and enables detection of 5-methoxyindoles in a pool of two hamster pineal glands. This is also the first time that these three 5-methoxyindoles have been measured simultaneously in plasma.

The concentrations of urinary phenylacetic, m- and p-hydroxyphenylacetic, mandelic (MA), p-hydroxymandelic, indoleacetic, homovanillic, vanillylmandelic, 5-hydroxyindoleacetic and 3,4-dihydroxyphenylacetic (DOPAC) acids and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and the concentrations of the above plasma acids (except MA, MHPG and DOPAC) were determined by gas chromatography-mass spectrometry with selected-ion monitoring at the time of receipt of the samples and after three, six and nine months storage at -18 degrees C. The samples were not treated in any way before storage. The concentrations of most of the urinary metabolites declined significantly (analysis of variance) over the nine-month storage period, whereas most of the plasma metabolites did not.

A method is described that allows determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in platelet-poor human blood plasma after direct injection of 50-100 microliters of plasma into a coupled-column liquid chromatographic system. The chromatographic system comprised an enrichment column and two separation columns with different selectivity properties. The samples were injected into the C18 enrichment column, which was eluted with a buffer solution as the mobile phase. 5-HT, 5-HIAA and the internal standard 5-hydroxy-N-methyl-tryptamine (5-HMT) were then desorbed by a stronger mobile phase and flushed into a cation exchanger, which separated 5-HT and 5-HMT. 5-HIAA passed straight through and was switched to a C18 column where it was retarded. After 5-HT and 5-HMT had been eluted from the cation exchanger and detected, the eluate from the C18 column was directed to the detector and 5-HIAA was determined. Basic plasma levels, about 4 nM for 5-HT and 30 nM for 5-HIAA, were measured with a relative standard deviation of about 5%.

A simple procedure for the simultaneous determination of major metabolites of dopamine and serotonin in plasma, i.e. 3,4-dihydroxyphenylacetic acid, 5-hydroxyindole-3-acetic acid, and 4-hydroxy-3-methoxyphenylacetic acid, was developed. The method is based on rapid isolation of the compounds by one-step clean-up on a small C18 column, followed by high-performance liquid chromatography with dual electrochemical detection. The system is readily used for clinical applications.

A rapid and sensitive method for the concurrent determination of 5-hydroxytryptophan, serotonin and 5-hydroxyindole acetic acid by reversed-phase high-performance liquid chromatography with electrochemical detection has been developed. The separation of the indolic compounds was achieved using a phosphate-citric acid eluent containing 5% methanol. Detection limits in the low picogram range were found. The method has been applied to the determination of the indolic compounds in rat and human brain tissues, as well as in human plasma and cerebrospinal fluid. Tissue and plasma preparation required only deproteinization before chromatography, while cerebrospinal fluid was directly applied to the column.

A method for high-performance liquid chromatographic separation and electrochemical detection of biogenic amines and metabolites in a variety of biological matrices is described. The method employs either homogenization, precipitation or dilution followed by direct injection of the samples and permits the chromatographic resolution of dopamine, norepinephrine, epinephrine, serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in brain; 3-methoxy-4-hydroxyphenylglycol, DOPAC, 5-HIAA and HVA in cerebrospinal fluid; 5-HIAA, HVA and 5-HT in plasma; and 5-HIAA and HVA in urine. Alterations in chromatographic conditions, voltammetry and in vivo pharmacological manipulations are employed to verify the identity of the putative neurotransmitter and metabolite peaks in the biological samples.

Sensitive methods for the determination of deuterated and non-deuterated indole-3-acetic acid and 5-hydroxyindole-3-acetic acid by combined capillary gas chromatography-negative-ion chemical ionization mass spectrometry were developed. Indole-3-acetic and 5-hydroxyindole-3-acetic acids were converted into pentafluorobenzyl and trifluoroacetylmethyl derivatives, respectively, after pre-purification by high-performance liquid chromatography. These derivatives were separated by gas chromatography and determined by selected ion monitoring. In the determinations, indole-3-acetic-2,2,2',4',5',6',7'-d7 acid and 5-hydroxyindole-3-acetic-3,3-d2 acid were used as internal standards. The methods developed in this work were used for the determination of deuterated and non-deuterated indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human urine samples collected before and after administration of L-tryptophan-3,3-d2.