Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine.

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C18 column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform-isopropyl alcohol (9:1, v/v) solution. Levels of 2.5.10(-8) M BE in urine (25 pmol/ml) could be determined.     

High-performance liquid chromatography with photochemical fluorimetric detection of tryptophan based on 4-fluoro-7-nitrobenzo-2-oxa- 1,3-diazole : Total protein amino acid analysis

The simultaneous determination of amino acids including trytophan is described. The NBD- F forms a single adduct with tryptophan as with other amino acids, but the adduct lacks intrinsic fluorescence. After ultraviolet irradiation, the adduct fluoresces (pale-green); the fluorescence intensity increases with increasing irradiation time at pH 2-10, Under the same conditions, the other amino acid adducts are slowly decomposed. When the tryptophan adduct, separated on a Nucleosil ODS column (150×4.6 mm, 6 μm), is irradiated in an on-line photochemical reactor (310 nm), its fluorescence peak appears between those of the phenylalanine and lysine adducts. The detection limit for tryptophan by the proposed method is 3 pmol; the limits for other amino acids are 10–100 fmol.     

Simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human urine by automated precolumn derivatization and semi-microbore column liquid chromatography with fluorescence detection.

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C18 semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-microl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.     

High performance liquid chromatography determination of cyanide in urine by pre-column fluorescence derivatization

A method for the determination of cyanide in human urine has been developed. The method is based on the reaction of cyanide with 2,3-naphthalenedialdehyde and taurine to give a fluorescent product for reversed-phase HPLC separation and fluorometric detection. After centrifugation followed by dilution of urine samples, the specimens could be analysed directly by this method. The recovery of cyanide added to urine at concentration levels of 50-1000 pmol/mL was 85-96%. The detection limit of cyanide was 30 pmol/mL in urine. The method was successfully applied to the analysis of urine from smokers and nonsmokers. The mean concentrations of cyanide were found to be 215 pmol/mL for the former and 84 pmol/mL for the latter.     

Analytical determination of specific 4,4′-methylene diphenyl diisocyanate hemoglobin adducts in human blood

4,4′-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4′-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography–high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population.     

High performance liquid chromatographic determination of 3 alpha, 5 beta-tetrahydroaldosterone and cortisol in human urine with fluorescence detection.

A simple and sensitive method for the simultaneous determination of 3 alpha, 5 beta-tetrahydroaldosterone (THALD) and cortisol in human urine is described. The method uses high performance liquid chromatography with fluorescence detection. THALD and cortisol, released by enzyme hydrolysis, and fludrocortisone (internal standard) are isolated by a Sephadex G-25M column and a Bond-Elut C18 cartridge, and then oxidized by cupric acetate to form the corresponding glyoxal derivatives. The glyoxal derivatives are converted into the fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene. The quinoxalines are successfully separated on a reversed phase column (L-column ODS) with isocratic elution and monitored fluorimetrically. The detection limits for THALD and cortisol are 0.45 and 1.18 ng/mL urine (0.65 and 2.65 pmol/100 microL injection volume), respectively, at a signal-to-noise ratio of 3 in a 100 microL injection volume. This method permits the precise and sensitive determination of THALD and cortisol in human urine (2 mL).     

High-performance liquid chromatographic determination of 3 alpha,5 beta-tetrahydroaldosterone in human urine with chemiluminescence detection.

A sensitive method for the determination of 3 alpha,5 beta-tetrahydroaldosterone (THALD) in human urine is described. The method uses high-performance liquid chromatography with chemiluminescence detection. Urinary THALD, released by enzyme hydrolysis, is isolated and concentrated using a Sephadex G-25M column and Bond-Elut C1 cartridges, and then oxidized by copper(II) acetate to form the corresponding glyoxal derivative. The glyoxal derivative is converted into the chemiluminescent quinoxaline by reaction with 4,5-diaminophthalhydrazide. The chemiluminescent quinoxaline is separated within 50 min on a reversed-phase column (TSKgel ODS-120T) with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the quinoxaline with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for THALD is 0.6 pmol (220 pg) ml-1 in urine [1.5 fmol (0.53 pg) per 20 microliters injection] at a signal-to-noise ratio of 3. This method permits the sensitive and precise determination of THALD in human urine (50 microliters) from normal subjects and a patient with primary aldosteronism.     

Quantitative detection of N(7)-(2-hydroxyethyl)guanine adducts in DNA using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography (HPLC) was combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to develop a sensitive and selective method for the quantitative measurement of N(7)-(2-hydroxyethyl)guanine (N(7)-HEG) adducts in DNA obtained from ethylene oxide-exposed biological samples. Selected reaction monitoring (SRM) was used as the detection mode while the fragmentation product ion at m/z 152 generated from the precursor protonated N(7)-HEG (m/z 196) was monitored. The detection limits for N(7)-HEG were estimated by twofold serial dilution and determined to be 4 fmol in neat standard solution and 16 fmol when a matrix effect is considered. When the mass spectrometer was operated in the selected ion monitoring mode using only the first quadrupole (without MS/MS function), the detection limits increased to 128 fmol and 1 pmol (when matrix effect is considered), respectively. A good linear correlation (R(2) = 0.999) was observed for signal intensities obtained by injecting 16 fmol--33 pmol of N(7)-HEG into the HPLC/ESI-MS/MS system. Hep G2 cells were incubated for 8 h with medium containing various concentrations of ethylene oxide (ranging from 0.05 to 5.0 mM). A dose-response relationship was established, indicating that the adduct formation increases with the exposure level. The method shows potential, although the detection limit needs to be lowered for practical applications, for use in monitoring N(7)-HEG formation in other biological systems.     

Separation and determination of homovanillic acid and vanillylmandelic acid by capillary electrophoresis with electrochemical detection

A method of separation and determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA) was developed based on capillary zone electrophoresis/amperometric detection with high sensitivity, good resolution and selectivity. In order to achieve complete separation and good response, several factors including pH, buffer concentration, separation voltage, detection potential and the length of separation capillary, were studied in detail. The method has been used to determine both HVA and VMA in human urine. Uric acid (UA) in human urine did not interference with their determination. The limit of detection of the method was 1.3×10⁻⁶ mol/l (1.4 fmol) for HVA and 7.9×10⁻⁷ mol/l (0.87 fmol) for VMA at a signal-to-noise ratio of 3.     

High-performance liquid chromatographic determination of formate as benzimidazole in biological samples

Formate was determined as benzimidazole by high-performance liquid chromatography after reaction with o-phenylenediamine at 130 degrees C for 2 h in 1 M perchloric acid. The useful concentration range was 1.6-40 mumol/l and the determination limit was 20 pmol. The recoveries from rat liver homogenate and human urine were 90.3 +/- 2.9 and 89.4 +/- 2.5%, respectively. Using this method, the activity of formaldehyde dehydrogenase in biological samples could be measured, and also the formate concentration in the liver and urine of rats to which methanol had been administered.     

A method for routine quantitation of urinary 8-hydroxy-2'-deoxyguanosine based on solid-phase extraction and micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.     

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.     

Simultaneous determination of 8‐oxo‐2′‐deoxyguanosine and 8‐oxo‐2′‐deoxyadenosine in DNA using online column‐switching liquid chromatography/tandem mass spectrometry

Sensitive and reliable methods are required for the assessment of oxidative DNA damage, which can result from reactive oxygen species that are generated endogenously from cellular metabolism and inflammatory responses, or by exposure to exogenous agents. The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) method is described, that utilises online column‐switching valve technology for the simultaneous determination of two DNA adduct biomarkers of oxidative stress, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) and 8‐oxo‐7,8‐dihydro‐2′‐deoxyadenosine (8‐oxodA). To allow for the accurate quantitation of both adducts the corresponding [¹⁵N₅]‐labelled stable isotope internal standards were synthesised and added prior to enzymatic hydrolysis of the DNA samples to 2′‐deoxynucleosides. The method required between 10 and 40 µg of hydrolysed DNA on‐column for the analysis and the limit of detection for both 8‐oxodG and 8‐oxodA was 5 fmol. The analysis of calf thymus DNA treated in vitro with methylene blue (ranging from 5 to 200 µM) plus light showed a dose‐dependent increase in the levels of both 8‐oxodG and 8‐oxodA. The level of 8‐oxodG was on average 29.4‐fold higher than that of 8‐oxodA and an excellent linear correlation (r = 0.999) was observed between the two adducts. The influence of different DNA extraction procedures for 8‐oxodG and 8‐oxodA levels was assessed in DNA extracted from rat livers following dosing with carbon tetrachloride. The levels of 8‐oxodG and 8‐oxodA were on average 2.9 (p = 0.018) and 1.4 (p = 0.018) times higher, respectively, in DNA samples extracted using an anion‐exchange column procedure than in samples extracted using a chaotropic procedure, implying artefactual generation of the two adducts. In conclusion, the online column‐switching LC/MS/MS SRM method provides the advantages of increased sample throughput with reduced matrix effects and concomitant ionisation suppression, making the method ideally suited when used in conjunction with chaotropic DNA extraction for the determination of oxidative DNA damage. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of a guanosine-malonaldehyde adduct in urine by high-performance liquid chromatography with a thiobarbituric acid reaction detector.

A new method for the determination of a guanosine-malonaldehyde adduct, beta-D-ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine-malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.     

Simultaneous determination of imidazoleacetic acid and N tau- and N pi-methylimidazoleacetic acids in human urine by high-performance liquid chromatography with fluorescence detection

A sensitive method for the simultaneous determination of urinary imidazoleacetic acid and N tau- and N pi-methylimidazoleacetic acids which employs high-performance liquid chromatography with fluorescence detection is described. The compounds were converted into the corresponding fluorescent esters by reaction with 4-bromomethyl-7-methoxycoumarin. These derivatives are separated by liquid chromatography on a Radial-Pak silica column. The detection limits for imidazoleacetic acid and N tau-and N pi-methylimidazoleacetic acids in urine are 15, 10 and 20 pmol/ml, respectively. The 24-h urinary excretion of imidazoleacetic acid and N tau-and N N pi-methylimidazoleacetic acids by healthy persons was 5.7-39.9, 4.3-24.6 and 1.5-19.3 nmol/mg of creatinine, respectively.     

Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography–high resolution mass spectrometry in mucosal biopsies of the lower esophagus

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3–2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 ± 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of esophageal mucosa (N = 14) yielding 5.6 ± 1.9 mg tissue and a mean adduct level of 6.13 ± 9.35 pmol HPB/mg DNA.     

UHPLC method for the simultaneous determination of β-blockers, isoflavones, and flavonoids in human urine

A simple method using solid-phase extraction (SPE) and ultra high-performance liquid chromatography (UHPLC) for the simultaneous determination of β-blockers, isoflavones, and flavonoids in human urine is developed. A statistical central composite design and response surface analysis is used to optimize the separation of the analytes. These multivariate procedures are efficient in determining the optimal separation condition using resolutions and retention time as responses. A gradient elution using a mobile phase consisting of 0.05% trifluoroacetic acid in water and acetonitrile is applied on a Hypersil GOLD column within a short analysis time of 4.5 min. UV detection was used to monitor the analytes. The suggested method was linear in a concentration range from 0.04-20.00 μg/mL, depending on the compound. The limits of detection ranged from 8.9 to 66.2 ng/mL. The precision was lower than 2.74%, and the accuracy was between 0.01-3.65%. The Oasis HLB column, with the highest recoveries, is selected for the pre-concentration step. This present paper reports, for the first time, a method for the simultaneous determination of β-blockers, isoflavones, and flavonoids in human urine samples. Furthermore, the developed method can also be applied to the routine determination of examined compounds concentrations in human urine.     

Simultaneous determination of angiotensins II and 1-7 by capillary zone electrophoresis in plasma and urine from hypertensive rats

We describe the procedure developed for the simultaneous detection and quantification of angiotensin II and angiotensin-(1-7), by capillary zone electrophoresis with UV detection by photodiode-array, at a wavelength of 200 nm, in the plasma and urine from hypertensive rats. Optimal separation was achieved with a 100 mM boric acid + 3 mM tartaric acid + 10 fM gold (III) chloride electrolyte solution at pH 9.80. The applied voltage was 30 kV and the capillary temperature was kept constant at 20 °C. The method was over the concentration range of 0.01-500 pmol/mL. All determination coefficients were higher or equal to 0.9985. Limits of detection and quantification for angiotensin II were 0.0110 pmol/mL (S/N = 3) and 0.0195 pmol/mL (S/N = 5), respectively. While for angiotensin-(1-7), the limits were 0.0112 pmol/mL (S/N = 3) and 0.0193 pmol/mL (S/N = 5), respectively. The present method offers a time-saving way to simultaneous determination of angiotensin II and angiotensin-(1-7), since it can be completed in 10 min, compared to other methodologies reported in the literature for capillary electrophoresis and liquid chromatography, which require more than 1 h for analysis of complex matrices, such as plasma and urine. The procedure is illustrated by experiments that quantify simultaneously angiotensin II and angiotensin-(1-7) in plasma and urine from hypertensive and normotensive rats, with and without antihypertensive treatment. The levels of angiotensin II and angiotensin-(1-7) detected in the experimental model, resulted in a recovery of 99.00-106.01% and a reproducibility of less than 10%. The proposed analytical method is a use full tool for the simultaneous detection of angiotensin II and angiotensin-(1-7) implicated in vascular remodeling in pathologies such as hypertension.     

High-performance liquid chromatography-electrochemical detection of 3-methylhistidine in human urine.

A reversed-phase high-performance liquid chromatographic method is described for the determination of 3-methylhistidine content in human urine using pre-column derivatization with phenylisothiocyanate, isocratic elution with 15 mM sodium acetate-acetonitrile (92:8, v/v) and electrochemical detection. The limit of quantitation was 0.1 pmol. The method has been applied in routine analyses of 3-methylhistidine in both clinical and research work.     

High-performance liquid chromatographic measurement of guanidino compounds of clinical importance in human urine and serum by pre-column fluorescence derivatization using benzoin

High-performance liquid chromatographic microanalyses for guanidino compounds in human physiological fluids have been accomplished by means of a pre-column fluorescence derivatization method using benzoin. The guanidino compounds in urine or deproteinized serum after ultrafiltration are converted to the fluorescent derivatives with benzoin in an alkaline medium, and the derivatives are separated simultaneously within 25 min on a reversed-phase column (mu Bondapak Phenyl) with a linear gradient elution of methanol in aqueous mobile phase (pH 8.5). The method permits the quantitative determination of guanidinosuccinic acid, methylguanidine, taurocyamine and guanidinobutyric acid at concentrations of as low as 8-78 pmol/ml in human urine and serum.