Determination of tryptophan and several of its metabolites in physiological samples by reversed-phase liquid chromatography with electrochemical detection

A new method for the concurrent assay of three tryptophan metabolites at the picomole level is described. The method has been developed for blood, urine, cerebrospinal fluid, and tissue samples such as whole brain, brain parts, and endocrine glands. Tryptophan itself, serotonin, and 5-hydroxyindoleacetic acid are isolated initially on extraction columns, eluted with a suitable solvent, and injected onto a liquid chromatograph with an amperometric detector. This general approach may be applicable to a variety of other tryptophan metabolites and should be useful in both research and clinical investigations.     

Quantitative estimation of salicylic acid and its metabolites by thin-layer densitometry

A rapid thin-layer densitometric method for the quantitative determination of salicylic acid and its metabolites in urine or plasma is described. The method is specific and very sensitive. Nanogram quantities of salicylic acid and its metabolites, both free and conjugated, may be estimated. Known metabolites, as well as the newly described gentisuric acid, were estimated quantitatively in urine from a patient treated with aspirin.     

High-performance liquid chromatographic assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid

A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.     

Simultaneous determination by GC-MS-SIM of o-, m-, p-hydroxyphenylacetic acid, 3:4-dihydroxyphenylacetic acid and homovanillic acid in biological samples using a common selected ion

A GC-MS-SIM method is described for the simultaneous determination of five acid metabolites, o-, m-, and p-hydroxyphenylacetic acid, 3:4-dihydroxyphenylacetic acid and homovanillic acid in biological samples using a common selected ion for all the acids. The TMS ethers of methyl or ethyl esters of these compounds have a common ion at m/e 179 which is used for their quantitation by selected ion monitoring. The molecular ion at m/e 238 of the three monohydroxy phenylacetic acids, which is also the M-30 ion for homovanillic acid, is also used for their quantitation. The GC conditions for their separation, the relative sensitivities and some advantages of TMS ethers over other derivatives are described. The application of this method to biological samples is illustrated by data on human urine and plasma.     

Simultaneous liquid chromatographic determination of vanillylmandelic acid, homovanillic acid, and 5-hydroxy-3-indoleacetic acid in urine, using isocratic elution and electrochemical detection

A method for the simultaneous measurement of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine is described. Based on reversed-phase liquid chromatography with electrochemical detection, the procedure employs isocratic elution, thus making it suitable for use in the less well-equipped clinical or research laboratory. A simple extraction of the acids from acidified urine into ethyl acetate, is followed by evaporating to dryness a portion of the organic layer, and redissolving the residue in chromatographic mobile phase. Up to 20 samples can be analysed in a single working day. The method is validated and the results obtained are compared with reference methods. The cause of contamination of the glassy carbon surface of the working electrode is investigated, and a simple electrochemical pretreatment is described that overcomes this problem. Finally, the extra clinical information that can be derived from multi-metabolite assays is considered.     

Simultaneous determination of urinary hippuric acid, o-, m- and p-methylhippuric acids, mandelic acid and phenylglyoxylic acid for biomonitoring of volatile organic compounds by gas chromatography-mass spectrometry

A sensitive and precise method for the simultaneous determination of hippuric acid, o-, m- and p-methylhippuric acids, mandelic acid and phenylglyoxylic acid, which are major urinary metabolites of toluene, o-, m- and p-xylenes, styrene and ethylbenzene, respectively, was developed. These metabolites were converted into their methyl ester derivatives with methanol in hydrochloric acid, and then quantitated by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring using a DB-1 capillary column. The injected compounds were quantitatively and reproducibly resolved within 19 min with a detection limit of 8-27 pg. The calibration curves were linear in the range of 0.05-25 μg for each compound, with correlation coefficients above 0.9999. This method was successfully used to analyze small amounts of both rat and human urine samples without any interference from coexisting substances. Overall recoveries of these compounds spiked in urine samples were 92-104%. The analytical results of the contents of these metabolites in the rat and human urine samples are presented.     

Method for the determination of gamma-L-glutamyl-L-dihydroxyphenylalanine and its major metabolites L-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the analysis of gamma-L-glutamyl-L-dihydroxyphenylalanine (gludopa) and its major metabolites L-dihydroxyphenylalanine (L-DOPA), dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is described. High sensitivity is achieved with a multi-cell coulometric detector utilising the specific electrochemical properties of gludopa (limit of detection 10 pg on-column). The retention time of gludopa was both pH-dependent and sensitive to negatively charged ion-pairing agents. An alumina-based solid-phase sample preparation technique with dihydroxybenzylamine as internal standard is described for plasma and urine (limit of detection 40 pg/ml) and an ultrafiltration technique is described for tissues (limit of detection 1-10 ng/g). After treatment with 50 mg/kg gludopa, in excess of twenty separate catecholic metabolic peaks can be detected in rat urine, whereas in humans after 9 mg/kg the only catechols detected were L-DOPA, dopamine and DOPAC.     

Simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene using high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

A simple and rapid method using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene in human urine specimens and standard solutions is described. A hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer was compared for the determination of metabolite of aromatic solvents in urine samples. The metabolites selected were: trans,trans-muconic acid, hippuric acid, o-, m- and p-methylhippuric acid and phenylglyoxylic acid. The compounds were well separated from each other on narrow-bore 1-mm i.d. reversed-phase LC C-18 columns. Average recoveries for loading 100 microL of urine samples varied from 88-110% and the quantification limits were less than 30 ng/mL for each analyte (3 ng/mL for trans,trans-muconic acid). The qualitative information obtained (mass accuracy, resolution and full-scan spectra) with the QqTOF mass spectrometer allows a secure identification of analytes in biological matrices.     

High-performance liquid chromatographic method for the determination of salicylic acid and its metabolites in urine by direct injection

A direct injection method has been developed for the determination of salicylic acid and its metabolites in urine. Urine samples are treated with hydroxylamine to convert salicyl acyl glucuronide to salicylhydroxamic acid, which can be accurately quantitated by direct injection into a high-performance liquid chromatographic system along with salicylic acid, gentisic acid and salicyluric acid. Salicyl phenolic glucuronide is quantitated by difference after hydrochloric acid hydrolysis at 65 degrees C with no loss of salicylic acid by sublimation or hydrolytic loss of salicyluric acid. This method has been applied to urine samples from human subjects and the results are discussed.     

Analysis of N-benzoyl-L-tyrosyl-p-aminobenzoic acid (bentiromide) metabolites in urine by ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of bentiromide metabolites in urine. The procedure involves no more than direct injection of the diluted urine sample, obviating the need for an extraction step or an internal standard. A mu Bondapak C18 column is used with a mobile phase of 0.01 M tetrabutylammonium chloride (pH 7.4)--methanol (9:1). A flow-rate of 1.4 ml/min, detection at 254 nm and column temperature of 40 degrees C are employed. These conditions were achieved by investigating the effects of mobile phase pH, and concentrations and types of organic modifiers, buffers and ion-pairing agents on the resolution of the metabolites. The analysis time is 18 min per sample and the coefficient of variation on replicate assays is less than 10% for most concentrations studied. Analytical recoveries were between 95 and 100% throughout the appropriate concentration ranges and no interferences were obtained with the exception of p-acetamidobenzoyl glucuronide which could be eliminated by treatment of the samples with beta-glucuronidase. Concentration profiles of the metabolites were studied in normal subjects, and the method was found to be potentially useful for clinical situations in which the existing bentiromide test leads to ambiguous results because of small bowel and hepatic dysfunctions.     

In vivo metabolism study of polygalic acid in rat using HPLC-ESI-MSn

A very simple and direct method has been established for the determination of polygalic acid and its metabolites in rat urine based on HPLC coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC-ESI-MS(n)). The rats were administered a single dose (100 mg/kg) of polygalic acid by oral gavage. The urine samples were collected and purified through a C(18) solid-phase extraction cartridge, and then these pretreated samples were injected into a reversed-phase C(18) column with a gradient elution program, whereas acetonitrile-0.5% aqueous formic acid was used as mobile phase and detected by an on-line MS/MS system. As a result, the parent drug and its four metabolites were identified and characterized in rat urine for the first time by comparing their changes in molecular mass (ΔM), retention times and full-scan MS(n) spectra with those of the parent drug. A possible metabolic pathway of polygalic acid was investigated and proposed. More importantly, the results demonstrated that the newly developed method (HPLC-ESI-MS(n)) was sensitive, simple and suitable for the determination of polygalic acid and its metabolites in biological samples.     

Determination of vanilmandelic acid in urine by coupled-column liquid chromatography combining affinity to boronate and separation by anion exchange

An automated liquid chromatographic method for assaying vanilmandelic acid in urine is described. Vanilmandelic acid and potential interfering substances, such as catechol compounds and their metabolites, have been tested for affinity to boronic acid-substituted silica at various pH values. Vanilmandelic acid and the internal standard, isovanilmandelic acid, were bound to the boronate matrix at an acidic pH, whereas for instance catecholamines were unretained and passed through the column. The alpha-hydroxycarboxylic acids were then desorbed by another mobile phase (pH 6.0) and transferred to an anion exchanger for chromatography and electrochemical detection. A relative standard deviation of 2.8% was obtained for the analysis of human urine samples containing 6.6 microM vanilmandelic acid.     

Direct concurrent measurement of urinary vanillylmandelic acid, 5-hydroxyindoleacetic acid and homovanillic acid by HPLC. Three methodologies compared

Three different direct HPLC methods for the determination of 3-methoxy-4-hydroxymandelic acid (VMA, vanillylmandelic acid), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylacetic acid (HVA, homovanillic acid) in urine were compared: two spectrofluorometric methods, applying discontinuous gradients, and one serial coulometric linear gradient method. The imprecision study (n = 6) revealed comparable coefficients of variation (CV), intra-assay ranging 1.4-11.1%, and inter-assay ranging 5.9-11.8% for physiological and moderately elevated levels of VMA, 5-HIAA and HVA. All methods showed good linearities up to 100 mumol/L for each of the three compounds studied. Analytical recoveries were 97-114% for VMA, 87-103% for 5-HIAA, and 80-95% for HVA. Recoveries were not dependent on urinary relative densities in the range 1.010-1.030 kg/L or on protein content (prior to acidification) in the range 0.1-3 g/L, or on the pH of conservation in the range 2-5 or on storage temperature in the range -20 - +22 degrees C for three weeks. The distributed-sample comparison revealed acceptable correlations and clinically unimportant accuracy differences between the methods. It is concluded that direct fluorometric and electrochemical HPLC methods can be used in the determination of major catecholamine and serotonin metabolites in human urine for clinical diagnosis and follow-up of neural crest and carcinoid tumours.     

Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry

Summary An analytical method for the simultaneous determination of the pyrethroid metabolites cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 g L^–1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.     

Simultaneous Ion-Pairing Liquid Chromatographic Determination of the Major Metabolites of Styrene and Carbamazepine,and of Unchanged Carbamazepine in Urine

Abstract

A high-performance liquid chromatographic method for a simultaneous quantitative determination of carbamazepine (CBZ) and of the major metabolites of CBZ (trans-10,11-dihydroxy-10,11-dihydrocarbamazepine, TDC; carbamazepine-10,11-epoxide, CBZ-E) and those of styrene (S) (hippuric acid, HA; mandelic acid, MA; phenylglyoxylic acid, PA) in the rat urine is described. Separation is achieved on a Nova-Pak reverse-phase column by isocratic elution. Excellent resolution was obtained by adding to the acetonitrile-water mobile phase, tetrabutylammonium chloride (0.005 M) and methanol (1%). Detection is effected by UV absorption at 230 nm with a total analysis time of less than 18 min. An aliquot of diluted urine is injected directly onto the liquid chromatographic column. The limits of sensitivity of CBZ, CBZ-E, TDC, HA, MA, and PA are 3.3, 2.0, 1.8, 3.1, 1.2, and 3.1 μg/ml of diluted rat urine, respectively. Precision and accuracy of the method are found to be acceptable. The method can be used for studying the interaction between these two xenobiotics. Preliminary studies have shown its potential application to human investigations.     

Quantitative determination of valproic acid and 14 metabolites in serum and urine by gas chromatography/mass spectrometry.

A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA-conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.     

High-performance liquid chromatographic determination of nicotinamide and its metabolites in human and murine plasma and urine.

A high-performance liquid chromatographic method is described which enables the determination of nicotinamide and eight of its possible metabolites in human and murine plasma and urine, using ion-pairing on a base-deactivated reversed-phase column. Calibration curves were linear up to 2 mumol/ml for nicotinamide and 200 nmol/ml for the metabolites; both the intra- and inter-assay relative standard deviations ranged between 1 and 8%. In murine plasma, the N-oxide was the major nicotinamide metabolite, but in man, formation of 1-methylnicotinamide and the 2- and 4-pyridones was also significant. In urine, nicotinuric acid was seen in the mouse, but no nicotinic acid metabolites were seen in man.     

Determination of uric acid and creatinine in human urine using hydrophilic interaction chromatography

Uric acid is the end-product of purine metabolism and a major antioxidant in humans. The concentrations of uric acid in plasma and urine are associated with various diseases and routinely measured in clinical and biomedical laboratories using enzymatic conversion and colorimetric measurement. In this study a hydrophilic interaction chromatographic (HILIC) method was developed for simultaneous determination of uric acid and creatinine, a biomarker of urine dilution and renal function, in human urine. Urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration. Uric acid and creatinine were separated from other components in urine samples and quantified using HILIC chromatography. A linear relationship between the ratio of the peak area of the standards to that of the internal standard and the concentration of the standards was obtained for both uric acid and creatinine with the square of correlation coefficients >0.999 for both analytes. The detection limits were 0.04 μg/mL for creatinine and 0.06 μg/mL for uric acid. The described HILIC method has proved to be simple, accurate, robust and reliable.     

Identification of the main metabolites of 2-ethylhexanoic acid in rat urine using gas chromatography-mass spectrometry.

The metabolites of 2-ethylhexanoic acid, an industrial chemical and the active ingredient in wood preservatives, were investigated in rat urine. Male Wistar rats were given 2-ethylhexanoic acid (2-EHA) in drinking water (600 mg/kg daily) for nine weeks, and then urine specimens were collected and analysed. The compounds were identified by gas chromatography-mass spectrometry in both electron-impact mode and chemical ionization mode. In addition to 2-EHA, ten different 2-EHA-related metabolites were found in the urine of 2-EHA-treated rats. The main metabolite was 2-ethyl-1,6-hexanedioic acid. Urine also contained 2-ethyl-6-hydroxyhexanoic acid and five other hydroxylated metabolites and two lactones, the detailed structures of which have not yet been elucidated. The unsaturated 5,6-dehydro-EHA was also identified; this is the metabolite corresponding to 2-n-propyl-4-pentenoic acid, the hepatotoxic metabolite of valproic acid. At least part of the 2-EHA is present in urine as a glucuronide conjugate.