Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The ¹²C-/¹³C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10 μL of urine. We believe that this method opens the possibility of generating a very comprehensive profile of the organic acid sub-metabolome that will be useful for comparative metabolomics applications for biological studies and disease biomarker discovery.     

尿中有机酸的毛细管气相色谱—质谱轮廓分析—用于苯丙酮尿症的诊断

应用毛细管气相色谱-质谱轮廓分析方法,测定了33例2.5～4.5岁健康儿童尿中有机酸种类及含量和8例拟诊为苯丙酮尿症儿童尿中的有机酸,结果表明患儿尿样中苯丙酮酸、苯乙酸、邻羟基苯乙酸、对羟基苯乙酸高于正常值10～470倍。为苯丙酮尿症的确诊提供了可靠方法。     

Carrier-free estimation of106Ru in urine

A simple carrier-free method has been developed for estimating 0.1 to 1.1 Bq of¹⁰⁶Ru in urine. The organic matter present in urine samples was completely destroyed by wet ashing using nitric acid-peroxide mixture in presence of sulfuric acid. Ruthenium was oxidized to tetroxide using potassium periodate and allowed to react in-situ with polyethylene powder. The powder was separated, washed, pelletized and its -activity was counted. Recovery of¹⁰⁶Ru is found to be better than 80% with a minimum detection limit of 0.2 Bq/dm³ which is less than the DRL value specified by International Commission on Radiological Protection (ICRP).     

Analysis of Glycocholic Acid in Human Plasma and Urine from Hepatocellular Carcinoma Patients

Glycocholic acid (GCA) has been identified as endogenous biomarker for hepatocellular carcinoma (HCC). To dissolve protein and liberate GCA from protein, ionic liquids (ILs) that contain chaotropic ions were used for pretreatment of liquid biological samples. Coupling with solid-phase extraction (SPE) and reversed-phase high-performance liquid chromatography (RP-HPLC), the novel sample pretreatment method was applied for quantitative determination of GCA in urine and plasma samples. Compared with the traditional organic solvents pretreatment of biological samples, the proposed method is “greener” and simpler, due to no use of volatile organic solvent, and avoiding centrifugation. Under the optimal conditions, when the GCA-free urine and plasma samples were spiked with GCA at 0.05–1.0 and 0.2–10 μmol L^?1, the recoveries of GCA ranged between 95.8–101.6 and 96.9–100.4%, respectively. These procedures only required 1 mL of urine and 3 mL of 3 mM ILs aqueous solution and 100 μL of plasma and 4 mL of 2 mM ILs aqueous solution, respectively. The proposed method has been successfully validated on a small sample size of 14 HCC patients and 14 healthy volunteers. For HCC patients, the mean concentration of GCA was 24.79 ± 6.86 and 31.98 ± 11.12 μmol L^?1 in urine and plasma samples, and was about 3 times and 24 times as much as in healthy people, respectively. The proposed method opens up a new possibility in early diagnosis of HCC.     

Preparation of no-carrier added 16-131I-hexadecanoic acid via halogen exchange: Comparison between two methods

A simple method for labelling of 16-Br-hexadecanoic acid (16-BrHDA) with radioactive iodine has been reported via nucleophilic¹³¹I-for-Br exchange in the dry state and in organic solvents. While preparation in some organic solvents such as acetic acid, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) required reaction times of more than 2 hours to give yields of 20–50%, halogen exchange in dry state at temperature of about 100 °C gave rise to yields of about 94% of radiochemically pure 16-¹³¹IHDA within 5 min. The labelling product could be purified by high performance liquid chromatography (HPLC) and was shown to be free of significant radiochemical impurities.     

Determination of Uric Acid in Human Urine by Ion‐exclusion Chromatography with UV Detection Using Pure Water as Mobile Phase

A simple, rapid and accurate ion‐exclusion chromatographic method coupled with a UV detector for the determination of uric acid in human urine samples has been developed. The separation was carried out on an ion‐exclusion column using only pure water as mobile phase. The detection wavelength was 254 nm and urine sample was injected directly without any pretreatment. Furthermore, the retention behavior of uric acid on the ion‐exclusion column was researched when pure water and 1 mmol·L^?1 HCl were used as mobile phase, respectively. The stability of uric acid was also further investigated within 28 days. In this method, the linear range of the calibration curve for uric acid was 0.25–100 mg·L^?1, and the detection limit calculated at S/N=3 was 0.02 mg·L^?1. The proposed ion‐exclusion chromatographic method has been used for the determination of uric acid in human urine.     

Determination of methylmalonic acid after diazonium derivatization by high-performance liquid chromatography

The use of a single prederivatization step in conjunction with high-performance liquid chromatography (h.p.l.c.) is described for the determination of methylmalonic acid (MMA). The method is based on the reaction of MMA and 4-diazobenzenesulfonic acid, which produces a derivative that has a molar absorptivity of about 9 × 10³ l mol^?1 cm^?1 at 353 nm. The derivatization reaction is optimized for various parameters. A reagent concentration of 3.3 mM at a reaction pH of 4.6 and a temperature of 100°C are optimal. The reaction product is separated from the excess of reagent and other interfering components by using a polystyrene-divinylbenzene column and a highly aqueous mobile phase. After a simple clean-up step, it is possible to quantify MMA in urine at about 0.8 mg l^?1 with linear response up to 32 mg l^?1.     

Quantitative determination of americium in biological materials

A simple, economic and specific radiochemical method for the determination of americium in urine and other biological materials is given. The urine sample is heated in nitric acid to break down any organic complexes of actinides. Insoluble phosphates are precipitated and changed to carbonates. The carbonate solution is passed through a diglycol succinate column and americium is eluted by hydrochloric acid. The eluate is evaporated to prepare a source for counting. The proposed procedure produces high overall yield and the sensitivity of the method is about 0.74 mBq (0.02 pCi). Determination of americium in the presence of other -emitters in urine is also given.     

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection--an efficient tool for monitoring of inborn metabolic disorders

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) and UV photometric detection. The optimized analytical system involved a 100 cm long polyacrylamide-coated capillary (50 μm i.d.) and the background electrolyte of 20 mM 2-morpholinoethanesulfonic acid (MES)/NaOH + 10% (v/v) methanol, pH 6.0 (pH is related to the 20 mM MES/NaOH buffer in water). The LOD values obtained by C⁴D for the OAs which do not absorb UV radiation range from 0.6 μM (oxalic acid) to 6.8 μM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9 μM (5-hydroxy-3-indoleacetic acid) to 10.2 μM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.     

Determination of urinary arsenic by electrothermal atomic absorption spectrometry with the l'vov platform and matrix modification

Total arsenic can be determined rapidly and simply in urine by dilution with a matrix modifier containing nickel and magnesium nitrate in nitric acid. The background correction capability of Zeeman-effect electrothermal atomic absorption spectrometry effectively nullifies nonatomic absorption by urine concomitants. Accuracy and precision of the method were evaluated by the determination of total arsenic in the National Bureau of Standards' SRM 2670, Toxic Elements in Freeze-Dried Urine, and commercially available lyophilized urine material. Deviations of determined concentrations from expected values ranged from about 4 to 18 ng ml^?1 with standard deviations ranging from about 5 to 31 ng ml^?1.     

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     

Enzymatic Fluorimetric Determination of Ursodeoxycholic Acid in Urine Using Clostridium Absonum 7β-Hydroxysteroid Dehydrogenase

Abstract

A simple and rapid enzymatic fluorimetric method for the determination of ursodeoxycholic acid (UDCA) and its glycine (GUDCA) and taurine (TUDCA) conjugates in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C₁₈) are used for the solid-phase extraction of bile acids (BA) from urine samples. the method is based on the fluorimetric monitoring of NADPH formed via the reaction of 7β hydroxylated BA (7β-BA) with β-nicotinamide adenine dinucleotide phosphate (β-NADP⁺) catalysed by 7β hydroxysteroid dehydrogenase (7β-HSD). the 7β-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC # 27555) and purified by affinity chromatography.

The method has a limit of detection of 2 μmol/L (initial sample concentration), within-run precision varied from 8.3% to 5.3% and between-run precision varied from 12% to 1.8% for low and high concentrations respectively. the recovery of ursodeoxycholic acid added to urine samples was about 98% (range 88–110%). the method was successfully applied for UDCA determination in urine samples from patients subjected to UDCA therapy. Randomly collected urine samples from patients and controls were used and the results were expressed as ratio of [UDCA]/[creatinine] to correct for variation in urine flow.     

Determination of a new blood glucose-lowering agent in biological fluids by capillary gas chromatography using electron-capture detection

A gas chromatographic method for the determination of 5-(4-chlorophenyl)-2-[(4-methylphenyl)sulphonyl]-4-pentynoic acid (I) in plasma (serum) and urine has been developed. After an extraction process, the cleaned-up organic extract was derivatized with diazomethane at ambient temperature. Results are evaluated from peak-height ratios with respect to the appropriate internal standard. The detection limit following extraction of a 1-ml plasma sample is about 20 ng/ml.     

A Novel Carbon Nitride Nanosheets-based Electrochemical Sensor for Determination of Hydroxychloroquine in Pharmaceutical Formulation and Synthetic Urine Samples

This study introduces modified carbon paste electrodes with carbon nitride nanosheets (CNNS) and outlines their application for the determination of hydroxychloroquine sulfate (HCQ) in tablets and synthetic urine samples. CNNS were synthesized by hydrothermal route (200 °C, 10 h) using melamine and citric acid as their precursors. The carbon nitride nanosheets-based electrode (CNNS/E) presented a linear dynamic range for HCQ (LDR), ranging from 10.0 nmol l⁻¹ to 6.92 μmol l⁻¹, and detection (LOD) and quantification limits (LOQ) of 0.16 nmol l⁻¹ and 0.52 nmol l⁻¹, respectively. LOD and LOQ were calculated by the equations: LOD=3(S_d/b), and LOQ=10(S_d/b). The modified sensor presented excellent relative standard deviations for parameters such as repeatability (2.39 % and 1.87 %) and reproducibility (3.22 % and 2.32 %) in HCQ oxidation peaks (1 and 2). The CNNS/E has not shown significant variations in its anodic signal intensity in the presence of some organic and inorganic substances. It is worth bearing in mind that CNNS/E can be easily manufactured and the sensor has the lowest HCQ detection limits reported so far. The proposed sensor was successfully applied for HCQ determination in tablets and synthetic urine, showing good recovery values and an error of 0.60 % about comparative method in tablet samples, assuring the quality of the method.     

The determination of bismuth in serum and urine by electrothermal atomic absorption spectrometry

A method for the determination of bismuth in serum and urine is presented. The method includes 1 + 1 dilution of the sample with 1 mM EDTA followed by charring in a graphite furnace under an oxygen atmosphere. The use of oxygen results in the formation of more homogeneous bismuth salts (probably bismuth oxides) during charring, and more efficient burning of the protein and organic components of the sample matrix. The procedure formulated is a standard additions method and is verified for serum and urine samples. The method reduces matrix interferences, gives a detection limit of 0.05 μg l^-1, and has a relative standard deviation of less than 5% for bismuth in the reference range of 2.6–6.0 μg l^-1. The method displays a recovery accuracy of 91, 96, and 100% for urine, serum, and aqueous samples, respectively.     

Liquid Chromatographic Determination of NSAIDs in Urine After Dispersive Liquid–Liquid Microextraction Based on Solidification of Floating Organic Droplets

An environmentally benign method of sample preparation based on dispersive liquid–liquid microextraction and solidification of floating organic droplets (DLLME-SFO) coupled with high-performance liquid chromatography with ultraviolet detection has been developed for analysis of non-steroidal anti-inflammatory drugs (NSAIDs) in biological fluids. A low-toxicity solvent was used to replace the chlorinated solvents commonly used in conventional DLLME. Seven conditions were investigated and optimized: type and volume of extraction solvent and dispersive solvent, extraction time, effect of addition of salt, and sample pH. Under the optimum conditions, good linearity was obtained in the range 0.01–10 µg mL⁻¹, with coefficients of determination (r ²) >0.9949. Detection limits were in the range 0.0034–0.0052 µg mL⁻¹ with good reproducibility (RSD) and satisfactory inter-day and intra-day recovery (95.7–115.6 %). The method was successfully used for analysis of diclofenac, mefenamic acid, and ketoprofen in human urine. Analysis of urine samples from a patient 2 and 4 h after administration of diclofenac revealed concentrations of 1.20 and 0.34 µg mL⁻¹, respectively.

     

A novel biamperometric biosensor for urinary oxalate determination using flow-injection analysis

A biosensor for determination of oxalate concentration in urine has been developed by immobilisation of oxalate oxidase and peroxidase on the surface of an interdigitated gold electrode. Enzyme immobilisation was performed using BSA and glutaraldehyde. Biamperometric measurements were made in flow conditions both in aqueous oxalate solutions (tested concentration range between 50 μM and 10 mM) and in real urine samples (tested measuring range between 5 and 100 μM). Optimal working conditions were examined for flow-injection analysis, and good correlation was achieved between added oxalate quantity and the one measured by biosensor in urine matrix (R² = 0.9983). The influence of some interferences (ascorbic acid, uric acid, paracetamol, acetylsalicylic acid) was also studied using biamperometric measurement mode.     

Determination of beryllium in urine by graphite-furnace atomic absorption spectrometry

A method has been developed for the determination of beryllium in urine by graphite-furnace atomic absorption spectrometry. Ammonium 12-molybdophosphate and ascorbic acid were employed as a matrix modifier. The tolerable charring temperature for beryllium in both aqueous solution and urine was raised to 1400 °C in the presence of a matrix modifier. The sensitivity for the determination of beryllium was also improved by a factor of 1.5 in comparison with that obtained by using magnesium nitrate as a matrix modifier. The mechanism of the enhancement effect of ammonium molybdophosphate was ascribed to the effectiveness of the formation of gaseous BeO, which is a precursor of free beryllium. Beryllium in urine can be determined simply by dilution with ascorbic acid solution. The relative standard deviation for seven replicate determinations of beryllium was 1.9% for a urine sample containing 0.029 μg ml^?1 of beryllium.     

Determination of plutonium in large volume of urine

A method was developed for the determination of plutonium in urine excreted within 72 hours (4–6 l) by an individual, mainly for monitoring internal contamination of persons working with plutonium. Plutonium is successively separated from organic and inorganic macrocomponents present in urine and from other alpha emitters by the help of coprecipitation techniques and by the help of extraction with TTA. The sample is measured on a ZnS scintillation detector. The lowest prbable activity is 0.15 pCi of²³⁹Pu/24 hrs urine.     

Carrier-mediated liquid phase microextraction coupled with high performance liquid chromatography for determination of illicit drugs in human urine

A new method was developed for the analysis of illicit drugs in human urine by coupling carrier-mediated liquid phase microextraction (LPME) to high performance liquid chromatography (HPLC). By adding an appropriate carrier in organic phase, simultaneous extraction and enrichment of hydrophilic (morphine and ephedrine) and hydrophobic (pethidine) drugs were achieved. Effects of the types of organic solvents and carriers, the carrier concentration in the organic phase, the HCl concentration in the acceptor solution, the stirring rate, and the extraction time on the enrichment factor of analytes were investigated. Under the optimal experimental conditions, high enrichment factors (202-515) were obtained. The linear detection ranges were 0.1-10 mg L⁻¹ for the studied drugs. The limits of detection (LOD) at signal-to-noise ratio of 3 were 0.05 mg L⁻¹ for both morphine and ephedrine, and 0.02 mg L⁻¹ for pethidine. This method was successfully applied to analysis of ephedrine in real urine specimens, revealing that the determination of illicit drugs in urine was feasible.