HPLC receptorassay of opioid peptides in the cerebrospinal fluid of lower back pain patients

Total opioid peptide receptoractivity in human cerebrospinal fluid is measured in patients who are experiencing lower back pain. Desalted CSF is eluted from a C18 Sep-Pak and is subjected to a radioreceptorassay (RRA) that employs tritiated etorphin, which is a ligand that is effectively displaced by opioids from several different types of opioid receptors. Three clinical groups have significantly different endogenous levels of 2.4, 4.5, and 6.4 pmol of methionine enkephalin-equivalents per mL CSF. Those three levels indicate that more opioid activity is correlated with the amount of drug to relieve the patient's perception of pain. When the total opioid content exceeds an empirical threshold, the sample is further fractionated with gradient reversed phase HPLC, and the opioid receptoractivity in each HPLC fraction is measured to determine the characteristic pattern of those receptoractive opioid peptides present in that patient's CSF. Different HPLC RRA patterns are found for different clinical categories. A possible interpretation of these two different sets of data.is that a lesion exists in one or several of the opioid peptidergic systems (metabolism, receptors) in this particular patient population.     

Proenkephalin A and proopiomelanocortin peptides in human cerebrospinal fluid

Precursors to beta-endorphin (BE) and methionine enkephalin (ME), and proteolytic enzymes that cleave those BE and ME precursors to BE and ME, were determined in several milliliters of human cerebrospinal fluid. Endogenous peptides were purified by reversed-phase high-performance liquid chromatography (HPLC), and were detected with radioreceptor assay (RRA), radioimmunoassay, and mass spectrometry (MS). Total opioid receptor activity measurements and the profile of HPLC-receptor activity of human CSF samples were both used to monitor neuropeptide metabolism. MS data linked the molecular ion of ME to a unique fragment ion. A later-eluting fraction (84 min) in a 90-min HPLC gradient appeared in all HPLC-RRA profiles, contained opioid receptor activity that displaced [3H]etorphine, and the quantitative and qualitative patterns of opioid receptor activity in those profiles both changed within the few minutes that elapsed between acquiring the first and second cerebrospinal fluid samples. That 84-min fraction contained precursors to opioid peptides and was fractionated further with a more shallow 120-min HPLC gradient into three sections that displayed delta-opioid receptor-preferring activity, using [3H]ME as ligand. These three sections were hydrolyzed separately with human cerebrospinal fluid as the source for endogenous neuropeptides to yield products that correlated to immunoreactive BE in section I and immunoreactive ME in section III.     

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.     

Enantiomeric determination of L- and D-lactic acid in human cerebrospinal fluid by chiral ligand exchange high-performance liquid chromatography

Enantiomeric determination of L- and D-lactate in human cerebrospinal fluid (CSF) was achieved by HPLC on a chiral stationary phase with UV detection. Samples were submitted to a solid-phase extraction procedure using Oasis HLB Plus Extraction Cartridge and L- and D-lactate in the extract were separated by Shodex ORpac CRX-453 B column, a ligand exchange column for chiral separation, using a mobile phase containing copper (II) ion. L- and D-lactate were determined in 25 min. Intra-assay precision in CSF was 4.98% (mean 1.85 mmol/L) for L-lactate and 10.1% (mean 4.96 micromol/L) for D-lactate (n = 5). Detection limits were between 1.0 (L-lactate) and 1.5 (D-lactate) pmol. The mean values (n = 3) of analytical recovery for L- and D-lactate were 95% and 107%, respectively. The mean +/- SD of concentrations of L- and D-lactate in CSF (n = 20) were 1.52 +/- 0.54 mmol/L and 10.98 +/- 5.15 micromol/L, respectively.     

High-performance liquid chromatographic determination of peptides released by tryptic degradation from opioid peptide precursors in rat brain.

A high-performance liquid chromatographic method involving postcolumn fluorescence derivatization is described for the quantification of five fragment peptides (methionine-enkephalin-Thr-Ser-Glu-Lys, methionine-enkephalin-Lys, methionine-enkephalin-Arg, leucine-enkephalin-Lys and leucine-enkephalin-Arg) released by tryptic digestion from the opioid peptide precursors (proopiomeranocortin and proenkephalins A and B) in rat brain tissues. The tissue proteins containing the precursors are hydrolyzed with trypsin to the fragment peptides. The peptides are separated on an Asahipak ODP-50 column and on-line detected fluorometrically by using hydroxylamine, cobalt(II) and borate buffer reagents. The detection limits (S/N = 3) for the peptides are 0.7-2.8 pmol per 100 microliters injected. The distribution of the precursors in the brain tissues was also discussed on the basis of the determined values of the fragment peptides.     

Isoform-specific quantification of endothelins in HUVEC culture supernatants by on-line high-performance liquid chromatography/electrospray mass spectrometry

A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform-specific and employs solid-phase extraction and subsequent HPLC fractionation followed by HPLC-ESIMS analysis. The peptide vasoactive-intestinal-contractor (VIC) was used as internal standard for the HPLC-ESIMS analysis. Linearity of calibration curves was from 50 fmol to 25 pmol. The limit of detection of the HPLC-ESIMS step using a buffer matrix was estimated at 50 fmol (S/N > 3). The overall limit of detection for supernatants of HUVEC was 500 fmol/mL. In HUVEC culture supernatants only ions of endothelin-1 (ET1) were observed. Basal levels were determined to be 1.8 +/- 0.3 pmol/mL. Quantitative results obtained for ET1 were in agreement with those obtained by using a standard addition method and by an ELISA method.     

HPLC with fluorescence detection of urinary phenol, cresols and xylenols using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a fluorescence labeling reagent.

A simple and sensitive HPLC method for the determination of phenolic compounds, i.e., phenol (Phe), cresols (Cres) and xylenols (Xyls), was developed. After a pre-column fluorescence derivatization of these compounds with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) at 60 degrees C for 30 min, 11 DIB derivatives were successfully separated within 50 min with an ODS column using CH3CN-H2O-CH3OH (25 + 22 + 53, v/v) as the eluent. The detection limits of DIB derivatives at a signal-to-noise ratio of 3 ranged from 0.15 to 1.09 microM (0.2-1.6 pmol per 20 microliters). The precision of the proposed method for both within- and between-day assays of free and total phenol related compounds was satisfactory (RSD < 9.5%). By the proposed method, Phe and p-Cre could be detected in normal urine samples, and the calculated concentrations of free Phe and p-Cre in unhydrolysed urine samples were 1.5 +/- 1.3 and 23.9 +/- 24.3 microM and those of total Phe and p-Cre in hydrolysed urine samples were 87.3 +/- 81.2 and 200.7 +/- 195.4 microM (n = 21), respectively.     

Pre-column 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.     

Study of transaldolase deficiency in urine samples by capillary LC-MS/MS

Transaldolase (TAL) is a key enzyme of the pentose phosphate pathway (PPP). TAL deficiency is a newly recognized cause of liver cirrhosis. We have developed an ion-pair LC separation combined with negative ion electrospray MS/MS detection method to assess PPP metabolites in urine samples from TAL-deficient mice. Sedoheptulose 7-phosphate (S7P), C5-polyols D-arabitol and D-ribitol, and 6-phosphogluconate (6PG) levels were markedly increased in urine of TAL-deficient mice with respect to those of wild-type and heterozygote littermates. The detection limits of S7P, D-arabitol, and 6PG were 0.15 +/- 0.015 pmol, 3.5 +/- 0.41 pmol, and 0.61 +/- 0.055 pmol, respectively. The limit of quantitation was 0.4 +/- 0.024 nmol/ml for S7P, 1.6 +/- 0.11 nmol/ml for 6PG and 10 +/- 0.7 nmol/ml for D-arabitol. Additional metabolites, hexose 6-phosphates (m/z 259), D-ribose 5-phosphate and D-xylulose 5-phosphate (m/z 229), D-fructose 1,6-diphosphate (m/z 339), C6-polyols (m/z 181) and GSSG (m/z 611), that have been positively identified in mouse urine, showed similar levels in control and TAL-deficient mice.     

Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.     

High-performance liquid chromatography/chemiluminescence determination of biological thiols with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide

The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH group. The labelling reaction was carried out at 60 degrees C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODS-80 column (150 x 4.6 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23 +/- 0.15 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.     

Determination of catecholamines and their 3-O-methyl metabolites in mouse plasma

The determination of catecholamines and their 3-O-methyl metabolites in a single mouse plasma is necessary to understand the role of the sympathetic nervous activity, while the inactivation of catecholamines by catechol-O-methyltransferase indicates the activity of blood pressure regulation in animals. Here we report the basal catecholamines and their 3-O-methyl metabolite concentrations obtained from 15 microL of mouse plasma utilizing semi-microcolumn high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence detection system. The concentrations were 6.63 +/- 1.37 pmol/mL plasma, 0.49 +/- 0.10 pmol/mL plasma, 5.25 +/- 2.30 pmol/mL plasma, 3.23 +/- 0.84 pmol/mL plasma, 0.44 +/- 0.11 pmol/mL plasma, and 3.39 +/- 1.67 pmol/mL plasma for norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively (n = 5-7). Further, when blood pressure was reduced by minoxidil, plasma catecholamines were found to be significantly increased by the baroreflex-mediated response in mouse.     

The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimer's disease: evidence of a novel carboxyterminally elongated Abeta peptide

The patterns of amyloid beta (Abeta) peptides in human cerebrospinal fluid (CSF) and brain homogenates were studied by surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry, and the results were compared with those obtained by Abeta-SDS-PAGE/immunoblot. Apart from the peptides known in the literature to occur in the CSF, we postulate the existence of a novel, previously not described peptide, either Abeta1-45 or Abeta2-46. This peptide was observed exclusively in a pool of samples originating from patients with AD, i.e. CSF and postmortem brain homogenates, but not in either the pooled CSF samples nor the pooled brain homogenates of the non-demented controls. Similarly to our previous results, Abeta1-42 was decreased in the CSF in AD. Expectedly, brain homogenates of the control subjects did not show the presence of Abeta peptides. Compared with Abeta-SDS-PAGE/immunoblot, SELDI-TOF enabled more precise analysis of Abeta peptides in the human material. We conclude that SELDI-TOF offers a promising tool for dementia expression pattern profiling using a minute amount of a biological sample.     

Study by means of high-performance liquid chromatography of solutes that decrease theophylline/protein binding in the serum of uremic patients.

Substantial changes in protein binding of drugs occur during the progression of renal insufficiency. Protein-bound uremic solutes play a role in the inhibition of drug protein binding. We previously demonstrated that hippuric acid in uremic ultrafiltrate was an inhibitor of the theophylline protein binding. The present study was undertaken to extend the yield of protein-bound uremic solutes by displacing ligands in uremic serum from their binding sites by five deproteinization methods. The inhibitory effect on theophylline protein binding of the deproteinized uremic serum was higher than with ultrafiltrate (p < 0.05). The influence of 30 semi-preparative HPLC fractions from deproteinized uremic serum on the theophylline protein binding was evaluated to identify the responsible compounds and to compare their relative individual impact. The theophylline protein binding was calculated as a percentage (bound versus total). The most important decrease of the protein binding was observed in HPLC fractions 6, 10 to 13, 15 and 28 with protein binding of: 61.5 +/- 10.8, 64.5 +/- 7.6, 60.9 +/- 10.1, 47.5 +/- 3.3, 60.0 +/- 6.7, 60.7 +/- 6.3 and 61.3 +/- 6.9%, respectively versus 69.1 +/- 2.4% for control serum (p < 0.05). The responsible compounds were characterized in the fractions by co-elution: 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), indole-3-acetic acid, indoxyl sulfate, hippuric acid, p-hydroxyhippuric acid and tryptophan. Their concentration was determined by analytical HPLC and a solution containing these compounds at the same concentration as in deproteinized uremic serum was composed. This solution was added to control serum and decreased the theophylline protein binding from 69.0 +/- 4.4% to 61.3 +/- 1.3%, which was less important than in genuine uremic serum (44.4 +/- 3.8%, p < 0.05). Dose-response curves with the characterized compounds revealed that the most important role in binding inhibition could be attributed to hippuric acid and CMPF. Our data suggests that the yield of protein binding inhibiting compounds is more important with deproteinized uremic serum than with uremic ultrafiltrate. The identified uremic compounds are not entirely representative for the decreased protein binding of theophylline, indicating that additional factors than those identified in this study affect the protein binding as well.     

Enzyme degradation, high performance liquid chromatography and liquid secondary ion mass spectrometry in the analysis of glycoproteins.

Analysis of small amounts of glycoproteins by high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS) together with enzyme digestion has been investigated using fetuin as a model. Preliminary data indicates that 71% of the expected peptides were detected by LSIMS analysis of 200 pmol total digest. HPLC profiles of peptides and glycopeptides were obtained from 2 nmol of digest using a reversed phase (C18) column eluted in a solvent system containing TFA, water and acetonitrile. This has provided glycopeptides for subsequent oligosaccharide analysis. Strategies are reviewed for the chromatographic characterization of oligosaccharides following their release from glycopeptides by chemical and enzymatic procedures.     

Determination of denzimol, a new anticonvulsant agent, and its main metabolite in biological material by gas chromatography and high-performance liquid chromatography

Two methods, using gas chromatography (GC) and high-performance liquid chromatography (HPLC), were developed in order to investigate the pharmacokinetics of denzimol hydrochloride, N-[beta-[4-(beta-phenylethyl)phenyl]-beta-hydroxyethyl] imidazole hydrochloride, which is a new anticonvulsant drug, and of its main metabolite, N-[beta-[4-(beta-phenyl-beta(alpha)-hydroxyethyl)phenyl] -beta-hydroxyethyl]-imidazole (referred to as M2), in humans. Both methods involve the use of a homologue of denzimol as an internal standard. The GC method is more sensitive (sensitivity limit 2.5 ng/ml for denzimol and 15 ng/ml for M2) and was utilized for the determination of denzimol and M2 in plasma. The GC method is specific, precise (relative standard deviations are 3.26, 2.12 and 1.72% at 10, 100 and 1000 ng/ml for denzimol and 6.45, 4.17 and 3.38% at 50, 500 and 1000 ng/ml for M2) and accurate (mean recovery +/- S.D. is 102.58 +/- 4.10% for denzimol and 99.72 +/- 7.75% for M2). The HPLC method is very simple and quick to perform. This method has a sensitivity limit of 0.5 micrograms/ml for denzimol and 1 microgram/ml for M2, and allows the determination of both compounds in urine with high selectivity, reproducibility (relative standard deviations are 2.05, 3.50 and 1.02% for denzimol and 2.78, 2.80 and 1.73% for M2, at concentrations of 15, 35 and 70 micrograms/ml) and accuracy (mean recovery +/- S.D. is 103.57 +/- 2.97% for denzimol and 95.91 +/- 1.59% for M2). The common anticonvulsants, when present in plasma, do not interfere with the monitoring of denzimol levels.     

Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography.

A simple, rapid and accurate method to separate and quantify cholesterol, desmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure is based on reversed-phase chromatography on a Inertsil ODS2 5 microm silica column with a binary gradient of mixtures of chloroform-methanol and chloroform-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterols are separated with good resolution and high reproducibility. The eluted sterols are quantified using a light-scattering (mass) detector. As little as 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, respectively, can be quantified under these conditions. Cholesterol is the predominant sterol both in spermatozoa (107+/-7 nmol/10(8) spermatozoa) and SP (0.83+/-0.10 micromol/ml) whereas the concentrations of desmosterol were 38+/-6 nmol/10(8) in spermatozoa and 0.18+/-0.02 micromol/ml in SP. Cholesterol sulfate represents about 6% of total cholesterol in the spermatozoa and SP. In conclusion, this method offers interesting perspectives for the quantitative analysis of these sterols not only in semen, but also in other biological samples.     

High-performance liquid chromatographic method with fluorescence detection for the determination of total homocyst(e)ine in plasma.

A high-performance liquid chromatographic method for the determination of total plasma homocyst(e)ine [H(e)] after reduction with sodium tetrahydroborate and precolumn derivatization with o-phthaldialdehyde is described. The analyses, carried out on a reversed-phase C18 column, were based on spectrofluorimetric detection. The sensitivity was 1 pmol per injection and the intra- and inter-assay relative standard deviations were 1.8% and 5%, respectively. The plasma H(e) concentration determined in 40 healthy volunteers (20-60 years old) was 12.4 +/- 2.9 microM (mean +/- S.D.), in good agreement with reference values.     

Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization

A high-performance liquid chromatographic method is described for the determination in human urine and plasma of catecholamines, their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine and L-DOPA), acidic compounds (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) and alcoholic compounds (3,4-dihydroxyphenylethyleneglycol and 4-hydroxy-3-methoxyphenylethyleneglycol)]. Urine (0.5 ml) containing 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid (internal standards) is deproteinized with perchloric acid, and the resulting solution is fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction), which include 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid, respectively. Plasma (0.7 ml) is deproteinized in the presence of 3,4-dihydroxybenzylamine (internal standard) in the same manner, and the resulting solution is directly used as an acid-alcohol fraction, while an amine fraction is obtained as for urine. Each fraction is subjected to the previously established ion-pair reversed-phase chromatography with post-column derivatization involving coulometric oxidation followed by fluorescence reaction with 1,2-diphenylethylenediamine. The detection limits, at a signal-to-noise ratio of 5, of the compounds measured in urine are 300 pmol/ml for the two mandelic acids, 2-7 pmol/ml for the other acidic and alcoholic compounds, 12 pmol/ml for L-DOPA and 0.6-2 pmol/ml for the other amino compounds; the corresponding values for plasma samples are 80, 0.5-3, 10 and 0.6-3 pmol/ml, respectively.