Liquid chromatographic determination of dicarboxylic acids based on intramolecular excimer-forming fluorescence derivatization

A highly sensitive and selective fluorimetric determination method for dicarboxylic acids (C5-C12) has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid hydrazide (PBH), followed by reversed-phase liquid chromatography (LC). The carboxylic acids were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acids. The structures of the derivatives and the emission of excimer fluorescence were studied by LC with mass spectrometry and with spectrofluorimetry, respectively. The PBH derivatives of the carboxylic acids could be separated by reversed-phase LC on an ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) were 1.3 fmol to undetectable for a 20-microl injection.     

Determination of methylmalonic acid in urine by HPLC with intramolecular excimer-forming fluorescence derivatization

We developed and validated an HPLC method with intramolecular excimer-forming fluorescence derivatization to determine methylmalonic acid, a unique biochemical marker for methylmalonic aciduria. Methylmalonic acid in urine and an internal standard were derivatized with pyrenebutyric hydrazide and separated on a C8 column. The derivatives were detected by monitoring the fluorescence at 475 nm (excitation wavelength 345 nm). At a signal-to-noise ratio of 3, the detection limit was 0.33 pmol on the column and the calibration curve was linear up to 1 mmol[sol ]L in urine. In a retrospective study on a relatively large number of known methylmalonic aciduria cases (n = 48), the method enabled us to differentiate methylmalonic aciduria cases from healthy controls (n = 52), regardless of age of patients at sampling or years of specimen storage. No interference was observed from isomeric or other dicarboxylic acids, or other urine constituents. As described, the method can be used retrospectively or prospectively for the diagnosis of methylmalonic aciduria and can be easily adopted by laboratories with no access to gas chromatography-mass spectrometry.     

Determination of catecholamines and indoleamines in human urine based on intramolecular excimer-forming derivatization and fluorescence detection.

A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-microL injection. Furthermore, this method was applied to a urine assay.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Determination of uranium in human urine by total reflection X-ray fluorescence

Uranium has been classified as a toxic chemical. It affects the kidneys, with nephritis being the primarily chemically-induced effect in animals and humans. Intermediate-term studies on animals indicate that increased uranium doses are positively correlated with various biochemical effects and histopathological changes. Since the kidneys efficiently excrete in urine the major portion of solubilized uranium circulating in blood, an increased urinary uranium excretion can provide a sensitive quantitative measure of exposure, especially in the case of acute exposure. In the present work a method was developed for the quantitative determination of uranium in human urine. It combines the chemical treatment of urine, which results in a significant pre-concentration of uranium, with its subsequent detection by means of total reflection X-ray fluorescence (TXRF). The method has been proven to be relatively fast, offering detection limits that allow for monitoring uranium intake above normal levels.     

Determination of trans-fatty acids in food samples based on the precolumn fluorescence derivatization by high performance liquid chromatography

Trans-fatty acids are unsaturated fatty acids that are considered to have health risks. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene is a highly sensitive fluorescent labeling reagent for carboxylic acids developed by our lab. In this study, using this precolumn fluorescent derivatization reagent, a rapid and accurate high-performance liquid chromatography with fluorescence detection method was developed for the determination of two trans-fatty acids in food samples. Under the optimized derivative conditions, two trans-fatty acids were tagged with the fluorescent labeling reagent in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide at 25°C for 30 min. Then, the baseline separation of trans- and cis-fatty acids and their saturated fatty acid with similar structures was achieved with less interference using a reversed-phased C₁₈ column with isocratic elution in 14 min. With fluorescence detection at λ_ex/λ_em = 490 /510 nm, the linear range of the TFAs was 1.0-200 nM with low detection limits in the range of 0.1–0.2 nM (signal-to-noise ratio = 3). In addition, the proposed approach was successfully applied for the detection of trans-fatty acids in food samples, and the recoveries using this method ranged from 96.02 to 109.22% with low relative standard deviations of 1.2–4.3% (n = 6).     

A novel method for determination of low molecular weight dicarboxylic acids in background atmospheric aerosol using ion chromatography

This paper describes a novel gradient elution ion chromatographic method using a Dionex AS11 system for the determination of low molecular weight dicarboxylic acids (low-M_w DCAs) in background atmospheric aerosol. Interference with the oxalic acid peak from sulfate in background PM_2.5 aerosol, 15.8 times the oxalic acid concentration, was remedied by removing sulfate using a barium cartridge, whilst interference with the malonic acid peak from carbonate was reduced by using a carbonate removal device. An alternative remedy to sulfate interference was use of an AS14 system using isocratic eluent, and this produced good resolution of oxalic acid from a high sulfate peak. In both the AS11 and the AS14 system, linear correlation coefficients were at all times >0.9990 with excellent linear range, the recoveries ranged from 92.8 to 106%, with relative standard deviation of 3.67-6.30%, whilst method detection limits (MDLs) ranged from 0.36 μg L⁻¹ for malic acid to 3.87 μg L⁻¹ for maleic acid. These data indicate that the analytical methods developed herein produce excellent separation efficiency and good determination of low-M_w DCAs with satisfactory accuracy, recoveries, and MDLs. Samples left at room temperature (20 °C) for 300 min in a simulation of the ‘waiting time’ involved in the proposed IC analysis decayed to between 86% (oxalic acid) and 39% (succinic and malonic acids) of their original concentration, whilst at 4 °C concentrations remained at 96-101% of original, indicating that maintaining samples at a low temperature prior to injection into the IC analyzer is vital for obtaining accurate results when analyzing low-M_w DCAs. Oxalic acid was found to be the most prevalent low-M_w DCA in background aerosol, comprising 57% of the total low-M_w DCAs and 0.959% of the PM_2.5 aerosol mass, followed by succinic acid and malonic acid.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: application to diagnosis of aminoacidopathies

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.     

Flow method based on cloud point extraction for fluorometric determination of epinephrine in human urine

A novel stepwise injection fluorometric method for the determination of epinephrine in human urine has been developed. In the current study, the stepwise injection analysis (SWIA) was successfully combined with on-line in-syringe cloud point extraction (CPE) and fluorometric detection. The procedure was based on the epinephrine derivatization in the presence of o-phenylenediamine followed by the preconcentration stage based on the CPE with the nonionic surfactant Triton X-114. After the phase separation into a syringe of the flow system, the micellar phase containing the epinephrine derivative was transported to a fluorometric detector. The excitation and emission wavelengths were set at 447 nm and 550 nm, respectively. The conditions of epinephrine derivatization and CPE have been studied. The calibration plot constructed using the developed procedure was linear in the range of 1·10⁻¹¹–5·10⁻⁷ mol L⁻¹. The limit of detection, calculated as 3 σ of a blank test (n = 10), was found to be 3·10⁻¹² mol L⁻¹. The proposed method was successfully applied for the determination of epinephrine in human urine samples.     

An improved derivatization method for sensitive determination of fatty acids by high-performance liquid chromatography using 9-(2-hydroxylethyl)-carbazole as derivatization reagent with fluorescence detection

Summary Tagging techniques with reagents used for fluorescent detection for short and long-chain fatty acids using high-performance liquid chromatography are evaluated in terms of the tagging reactions, handing, flexibility, stability of the reagents. Emphasis is given to the applications of the tagging techniques to relatively high molecular mass fatty acids. The fatty acids or carboxylic compounds were derivatized to their corresponding esters with 9-(2-hydroxy ethyl)-carbazole (HEC) in acetonitrile at 60°C with N, N′-carbonyldiimidazole (CDI) as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C₁−C₂₀ fatty acids was completely separated with 45 min using gradient elution on a reversed-phase C₁₈ column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (λ_ex 293 nm). Studies on derivatization conditions indicated that fatty acids react rapidly and smoothly with HEC in the presence of CDI and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The relative standard deviations (n=6) for each fatty acid derivative are <5.0%. The detection limits are at 38–57 fmol levels for C₁₄−C₂₀ fatty acids and lower levels for <C₁₄ fatty acids.     

A high performance liquid chromatography-electrochemical array method for the measurement of oxidative/nitrative changes in human urine

Oxidative stress has been implicated in various pathologies as well as in environmental pollutant-induced negative health outcomes. In this work we have developed an analytical method to measure oxidative stress markers namely m-, o-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine, and the DNA damage marker 8-hydroxy deoxyguanosine in human urine. The method involves the base hydrolysis of the urine sample followed by solid phase extraction of target analytes using a reverse phase polymeric sorbent column prior to the HPLC-EC analysis. The recovery studies indicated that the overall method recovery for all analytes were >60%. The limit of quantification of all target analytes was <10 nM with a linear range from 2 nM to 150 μM. The applicability of this method is demonstrated by analyzing the above markers in healthy human urine (n = 10) samples.     

A rapid HPLC method for the determination of carboxylic acids in human urine using a monolithic column

A rapid HPLC method for the determination of carboxylic acids in urine samples using a Chromolith Performance RP/18e 100/4.6 with Chromolith Guard Cartridge RP/18e 10/4.6 (Merck KgaA, Darmstadt, Germany) was developed. The method facilitates the simultaneous determination of aromatic hydrocarbon metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene, hippuric acid (HA) from toluene and 2-, 3-, 4-methylhippuric acids (MHA) from xylene. 3-Hydroxybenzoic acid (3-HBA) was used as internal standard. A chromatographic run is completed within less than 5 min for styrene, ethylbenzene and toluene metabolites, and within 10 min for xylene metabolites. The detection limits are 9 mg L^–1 urine for MA, 1.25 mg L^–1 urine for PGA, 4.9 mg L^–1 urine for HA, 22 mg L^–1 urine for 2-MHA, and 18.5 mg L^–1 urine for 3-MHA.No significant differences of the MA, PGA and HA concentrations in human urine samples obtained by HPLC chromatography on LiChrosorb RP 18 and on Chromolith RP/18e columns were found. The results were evaluated by using ANOVA.Abbreviations MA mandelic acid - PGA phenylglyoxylic acid - HA hippuric acid - MHA methylhippuric acid - 3-HBA 3-hydroxybenzoic acid - ANOVA analyses of variance - GC gas chromatography - HPLC high-performance liquid chromatography     

A multivariate multianalyte screening method for sulfonamides in milk based on front-face fluorescence spectroscopy

Screening methods are used to detect the presence of a substance or class of substances at the level of interest and are specifically designed to avoid false compliant results. They should allow the running of a high number of samples per day at a low cost under routine conditions. In this work, a rapid and simple method for the screening of six sulfonamides (sulfadiazine, SD; sulfamerazine, SMR; sulfamethazine, SMT; sulfachloropyridazine, SCP; sulfathiazole, STZ and sulfamethoxazole, SMO) in milk samples is proposed and assessed according to the criteria required by the European Regulation, Decision 2002/657/EC. The method is based on modelling front-face fluorescence emission spectra by means of partial least squares class modelling (PLS-CM). The milk samples are pre-treated with a single easy step of derivatization with fluorescamine.After confirming that the method has equal analytical sensitivity for all the six sulfonamides, it is established that the multivariate analytical sensitivity at 100 μg L⁻¹ is 37.5 μg L⁻¹ when analysing a mixture of six sulfonamides added to different brands of milk and measured in different days. In addition, the method is applied to samples from 11 commercial brands of milk. For β = 0.05, threshold value established by the Decision 2002/657/EC for this method, the probability of false non-compliance, α, is equal to 0.17, allowing the suitable screening of these six sulfonamides.     

Development of an HPLC method for the determination of mexiletine in human plasma and urine by solid-phase extraction

A sensitive and specific high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of mexiletine (MEX) in human plasma and urine. It uses solid-phase extraction (SPE) followed by an automated reversed-phase HPLC with a pre-column derivatization with 4-chloro-7-nitrobenzofurazan (NBD-CI) and UV-vis Absorbance detection. The process was set as: the UV-vis Absorbance wavelength was set at 458 nm. Chromatographic separation was performed on a Phenomenex-C₁₈ Column (Aqua, 150 mm × 4.6 mm i.d. with 5 μm particle size) with the mobile phase consisting of acetonitrile and water (80:20, v/v), and the flow rate was set at 1.0 mL min⁻¹. Calibration of the overall analytical procedure gave a linear signal (r > 0.9998) over a MEX concentration range of 0.2-2.0 μg mL⁻¹ in human plasma and urine. The detection limit in plasma and urine was 0.1 μg mL⁻¹. Intra- and inter-day precision of the assay at three concentrations within this range were 0.31-2.50%. The high specificity and sensitivity have been achieved by this fast method (total run-time <6 min). The method has been successfully validated in human plasma and urine and it has been shown to be precise, accurate and reliable.     

A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.     

Development of a method for the determination of advanced glycation end products precursors by liquid chromatography and its application in human urine samples

A liquid chromatographic method with fluorimetric detection has been developed to determine the most abundant α‐dicarbonyl compounds, generated as intermediates in the Maillard's reaction, previous derivatization to high fluorescent pteridinic derivatives. Hence, the biomarkers D‐glucosone, 3‐deoxyglucosone, glyoxal, methylglyoxal, diacetyl, 2,3‐pentanedione, and phenylglyoxal were quantified using a gradient elution mode. The experimental conditions of the derivatization reaction and mobile phase composition were optimized. Linearity ranges (peak area versus α‐dicarbonyl compound concentration) from 1.0 to 100.0 ng mL^?1 were obtained. Detection limits were comprised between 0.3 and 11.0 ng mL^?1. The high sensitivity of the method allows the determination of α‐dicarbonyl compounds present in human urine, such as D‐glucosone, 3‐deoxyglucosone, glyoxal, and methylglyoxal, that are used as biomarkers, in order to investigate their roles in several diseases, with special emphasis in diabetes mellitus. With the aim of avoiding the interferences due to pteridinic compounds present in urine, a cleanup step with an ISOLUTE ENV+ cartridge was carried out. The concentrations of these urinary biomarkers have been reported as a normalized ratio to urinary creatinine, and determined in healthy and in diabetic volunteers, of different ages and sex. In all urine samples, standard addition and external calibration procedures were applied and compared.     

An HPLC method for the determination of selected amino acids in human embryo culture medium

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra‐cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at −80°C. Filtered medium samples were derivatized with ortho ‐phthalaldehyde (naphthalene‐2,3‐dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse‐phase columns (LichroCART, Purospher STAR RP_18e or Ascentis Express C₁₈) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra‐assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.     

A sensitive method for simultaneous determination of four macrolides by CE with electrochemiluminescence detection and its applications in human urine and tablets

A sensitive method for simultaneous determination of azithromycin (AZI), acetylspiramycin (ACE), erythromycin (ERY), and josamycin (JOS) was developed by CE coupled with electrochemiluminescence detection with Ru(bpy)₃²⁺. The parameters related to separation and detection were investigated in detail. The four macrolides were well separated and detected within 6 min under the optimized conditions. The LOD (S/N=3) of AZI, ACE, ERY, and JOS were 1.2×10^?9, 7.1×10^?9, 3.9×10^?8 and 9.5×10^?8 mol/L, respectively. The LOQ (S/N=10) of AZI, ACE, ERY, and JOS in human urine were 8.2×10^?8, 2.5×10^?7, 8.9×10^?7 and 1.2×10^?6 mol/L, respectively. The recoveries of the four macrolides in human urine and pharmaceutical tablet samples were 85.0–104.0% at different concentration levels.     

A new flow injection preconcentration method based on multiwalled carbon nanotubes for the ETA-AAS determination of Cd in urine

A new flow injection (FIA) procedure for the preconcentration of cadmium in urine using multiwalled carbon nanotubes (MWCNT) as sorbent and posterior electrothermal atomization atomic absorption spectrometry (ETA-AAS) Cd determination has been developed. Cadmium was retained in a column filled with previously oxidized MWCNTs and it was quantitatively eluted with a nitric acid solution. The parameters influencing the adsorption-elution process such as pH of the sample solution, amount of sorbent and flow rates of sample as well as eluent solutions have been studied. Cd concentration in the eluent was measured by ETA-AAS under the optimized conditions obtained. The results indicated the elimination of urine matrix effect as a consequence of the preconcentration process performed. Total recovery of cadmium from urine at pH 7.2 using a column with 45 mg of MWCNTs as sorbent and employing a HNO₃ 0.5 mol L⁻¹ solution for elution was attained. The detection limit obtained was 0.010 μg L⁻¹ and the preconcentration factor achieved was 3.4. The method showed adequate precision (RSD: 3.4-9.8%) and accuracy (mean recovery: 97.4-100%). The developed method was applied for the determination of cadmium in real urine samples from healthy people (in the range of 0.14-2.94 μg L⁻¹) with satisfactory results.