Automated sample treatment with the injection of large sample volumes for the determination of contaminants and metabolites in urine

This work reports the development of a simple and automated method for the quantitative determination of several contaminants (triazine, phenylurea, and phenoxyacid herbicides; carbamate insecticides and industrial chemicals) and their metabolites in human urine with a simplified sample treatment. The method is based on the online coupling of an extraction column with RP LC separation–UV detection; this coupling enabled fast online cleanup of the urine samples, efficiently eliminating matrix components and providing appropriate selectivity for the determination of such compounds. The variables affecting the automated method were optimized: sorbent type, washing solvent and time, and the sample volume injected. The optimized sample treatment reported here allowed the direct injection of large volumes of urine (1500 μL) into the online system as a way to improve the sensitivity of the method; limits of detection in the 1–10 ng/mL range were achieved for an injected volume of 1500 μL of urine, precision being 10% or better at a concentration level of 20 ng/mL. The online configuration proposed has advantages such as automation (all the steps involved in the analysis – injection of the urine, sample cleanup, analyte enrichment, separation and detection – are carried out automatically) with high precision and sensitivity, reducing manual sample manipulation to freezing and sample filtration.     

Electrochemical evaluation of the desloratadine at bismuth film electrode in the presence of cationic surfactant: Highly sensitive determination in pharmaceuticals and human urine by Linear sweep-cathodic stripping voltammetry

In this study, the electrochemical properties of desloratadine, which is in the second generation antihistamines group, were determined by bismuth film electrode (BiFE) in aqueous and aqueous/surfactant solutions. This compound gave an irreversible and diffusion-controlled reduction peak at about –1.65 V by cyclic voltammetry. It was found that the addition of cationic surfactants (cetyltrimethylammonium bromide (CTAB) increased the reduction current signal of desloratadine, while anionic (sodium dodecylsulfate (SDS) and nonionic (Tween 80) surfactants were found to have an adverse effect. Using linear sweep-cathodic stripping voltammetry, the analytical signal showed a linear correlation with a concentration of 0.1 to 4 µM in 0.04 M Britton–Robinson solution (pH = 8.0) in the presence of 5 mM CTAB, while the detection limit was calculated to be 11.70 nM (3.64 μgL^–1). This method has been successfully applied for the quantitation of desloratadine in pharmaceutical and urine samples without the need for any separation.     

A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

Electrochemical reduction of cefminox at the mercury electrode and its voltammetric determination in urine

The electrochemical behaviour of cefminox in phosphate buffers solutions over pH range 2.0-9.0 using differential-pulse polarography, DC-tast polarography, cyclic voltammetry and linear sweep voltammetry (staircase) has been studied. In acidic media, a non reversible diffusion-controlled reduction involving two electrons occurs and the mechanism for the reduction was suggested. A differential-pulse polarographic method for the determination of cefminox in the concentration range 5.8×10⁻⁶-6.0×10⁻⁵ M with a detection limit of 1.76×10⁻⁶ M was proposed. Also, a method based on controlled adsorptive pre-concentration of cefminox on the hanging mercury drop electrode followed by linear sweep voltammetry, allows its determination in the concentration range 8.3×10⁻⁸-1.5×10⁻⁶ M with a detection limit of 2.47×10⁻⁸ M. These methods have been used for the direct determination of cefminox in human urine with recoveries between 98 and 103%, and precision around ±2%.     

Rapid and sensitive determination of strychnine and brucine in human urine by capillary electrophoresis with field‐amplified sample stacking

A simple, rapid, sensitive and low‐cost method using capillary electrophoresis (CE) coupled with field‐amplified sample stacking (FASS) has been developed and validated for the simultaneous determination of strychnine and brucine residues in human urine. Before sample loading, a water plug (3.5 kPa, 3 s) was injected to contain sample cations and to permit FASS. Electrokinetic injection at a voltage (20 kV, 25 s) was then used to introduce cations. Separation was performed using 20 mM acetate buffer (pH 3.8) with an applied voltage of 20 kV. The calibration curves were linear over a range of 8.00–2.56 ∞ 10² ng/mL (r = 0.9995) for strychnine and 10.0–3.20 × 10² ng/mL (r = 0.9999) for brucine. Extraction recoveries in urine were greater than 79.6 and 82.8% for strychnine and brucine, respectively, with an RSD of less than 4.9%. The detection limits (signal‐to‐noise ratio 3) for strychnine and brucine were 2.00 and 2.50 ng/mL, respectively. A urine sample from one healthy female volunteer (26 years old, 50 kg) was pretreated and analyzed. Strychnine and brucine levels in urine could be detected 24 h after administration. On these grounds, this method was feasible for application to preliminary screening of trace levels of abused drugs for both doping control and forensic analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Automated on-line HPLC-HRGC: Instrumental aspects and application for the determination of heroin metabolites in urine

A fully automated on-line HPLC-HRGC instrument is described. Samples are loaded into an HPLC autosampler. Pre-separation is carried out, automatically transferring the previously determined HPLC fraction to GC. Total HPLC fractions are introduced into GC, using the on-column or the loop-type interface, depending on the solvent evaporation technique applied. The HPLC column is automatically backflushed with a suitable solvent during GC analysis. The instrument was used for analyzing heroin metabolites, particularly morphine, in urine samples. Raw urine extracts were injected into HPLC and analyzed by GC using FID.     

Molecularly imprinted solid-phase extraction for the selective HPLC determination of ractopamine in pig urine

A method was developed for the determination of ractopamine in pig urine using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography. The molecularly imprinted polymer (MIP) was synthesized in acetonitrile-triethylamine system using ractopamine (RAC) as the template and acrylamide as the monomer. The binding capacity of the polymer toward RAC was found to be about 2.57 mg of ractopamine/g of polymer. The optimal procedures for MISPE consisted of conditioning with 3 mL methanol, equilibrating with 3 mL of water, loading volume of <10 mL of aqueous sample (pH 7), washing with 3 mL water and 3 mL methanol, and eluting with 5 mL of 5% ammonia in methanol. In the four spiked samples with the levels of 0.01, 0.1, 1.0 and 5.0 μg/mL, the mean recoveries of analyte on the MIP were higher than 90% with relative standard deviation <10%, and significant differences between imprinted and non-imprinted materials were observed. The MIP selectivity was evaluated by checking 11 drugs with similar and different molecular structures to that of RAC. The characteristics of three-dimensional cavities and hydrogen bond interaction were regarded as the main factors that dominated the retention of RAC on the MISPE cartridge.     

Bowl-shaped hollow N-doped carbon as an electrochemical platform for the highly selective and sensitive electrochemical detection of isoprenaline in pharmaceutical injection

In this study, bowl-shaped N-doped hollow carbon sphere-containing mesoporous nanomaterials (BNHC) were used for the ultrasensitive electrochemical detection of isoprenaline (ISOP). The linear ranges of the BNHC-based sensor were as follows: 0.05 – 15 μM; 15 – 70 μM, with the limit of detection (LOD) of 6.8 nM. And the BNHC-based electrochemical sensor showed a good current response to ISOP and maintained 95.6 % of the initial current after 30 days of storage. Notably, when the BNHC-modified sensor was used to detect the actual ISOP injection and urine samples, the sensor exhibited satisfactory recovery.     

Field‐amplified sample injection combined with CE for the enantiodetermination of cathinones in urine samples

This work presents a capillary electrophoresis methodology for the enantiodetermination of cathinones in urine employing a liquid–liquid extraction sample pretreatment. The cathinones were enantioseparated by adding a mixture of 8 mM 2‐hydroxypropyl β‐cyclodextrin and 5 mM β‐cyclodextrin to the background electrolyte, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Field‐amplified sample injection was used as preconcentration strategy to improve the sensitivity. We studied various parameters that affect this stacking strategy, in particular, the sample solvent and its pH, the presence or absence of a low conductivity solvent plug introduced before the sample injection, the nature and volume of this plug, and the voltage and time of the electrokinetic injection of the sample. The optimum conditions were achieved by injecting a plug of isopropanol:H₂O 50/50 at 50 mbar for 5 s prior to the electrokinetic injection of the sample prepared in an aqueous solution of HCl 10^?6 M. The sensitivity enhancement factors were from 562 to 601 in terms of peak area and from 444 to 472 in terms of peak height. The method was validated by analyzing spiked urine samples, obtaining a linear range of 25 to 1000 ng/mL and limits of detection ranging from 15 to 45 ng/mL.     

Evaluation of a spectrofluorimetric method for the selective determination of thalidomide in pharmaceutical tablets, urine and blood serum

A spectrofluorimetric analytical methodology for the determination of thalidomide in pharmaceutical formulations, blood serum and urine was developed and evaluated in terms of sensibility and selectivity. Efforts were spent on the maximization of thalidomide fluorescence signal and on the increasing of selectivity when analyzing complex samples, specially, in samples containing sulfanilamide, a strongly fluorescent concomitant that can be used in association with thalidomide. Maximum signal was observed with optimized composition of the solvent system and pH. Approaches to enable selective determination of thalidomide in the presence of sulfanilamide were tested and compared. Better results were achieved using the combined use of acidic medium and UV treatment. For urine and blood serum samples, a solid phase extraction (on a C₁₈ cartridge) was found to be practical, enabling good recovery results. Limit of detection (3S_b/m) was estimated to be 1.2 μg l⁻¹, approximately 100 times better than the ones reported for the routine UV absorption HPLC methods. This low-cost spectrofluorimetric methodology was found to be very simple and enabled excellent recoveries when analyzing analyte spiked biological fluids as well as thalidomide based commercial and laboratory made pharmaceutical formulations.     

Piezoelectric quartz crystal sensors based on ion-pair complexes for the determination of cinchonine in human serum and urine

A new sensing method for the determination of cinchonine was presented, based on the sensitive mass response of PQC and the selective adsorption of cinchonine on PVC membrane containing ion-pair complex. The proposed sensors show much wider working range (pH range), much higher sensitivity and selectivity than those potentiometric and spectrophotometric methods. In this paper, three counter ions were compared and six complexes synthesized. For the sensor modified with cinchonine-monotetraphenylborate-PVC membrane, calibration graph was linear over concentration range of 5×10⁻⁸-2.1×10⁻⁴ M with a detection limit of 4×10⁻⁸ M at pH 7.1, and recoveries 97.7-108.6%. Influencing factors were investigated in detail and optimized. Results from real samples were satisfactory.     

Simultaneous determination of guanosine and guanosine-5'-triphosphate in biological sample using gold nanoparticles modified indium tin oxide electrode

A nanogold modified indium tin oxide (ITO) electrode was used for the simultaneous determination of guanosine and GTP at pH 7.2. The electrode exhibited an effective catalytic response towards their oxidation and lowered the oxidation potential of guanosine by ∼120 mV and GTP by ∼183 mV. Linear concentration curves were obtained for guanosine with a detection limit of 9.8 × 10⁻⁸ M and 5.5 × 10⁻⁸ M for GTP. The concentration of guanosine and GTP were also estimated in the human blood plasma samples using gold nanoparticles modified ITO electrode with good reproducibility.     

Development of a selective sample clean-up method based on immuno-ultrafiltration for the determination of deoxynivalenol in maize

This paper describes the development of a highly selective analytical method for the determination of deoxynivalenol (DON) in maize. The developed method is based on immuno-ultrafiltration (IUF) and is the first application of IUF as a clean-up strategy in food analysis. Quantification of DON was carried out by high-performance liquid chromatography with ultraviolet detection. In contrast to immunoaffinity chromatography, in IUF the antibodies are not bound to a solid support material but used in free form, thus making it possible to avoid the critical immobilisation step. Sample clean-up by IUF proved to be as selective as clean-up using commercially available immunoaffinity columns. The limit of detection (S/N=3) of the analytical method was found to be 74 ng DON/g maize. Repeated analysis of a certified maize reference material on four different days resulted in a mean recovery of 93% with a standard deviation of 10%.     

Use of a diazocoupling reaction for sensitive and selective spectrophotometeric determination of furosemide in spiked human urine and pharmaceuticals

Two simple, sensitive, and selective spectrophotometric methods for the determination of 5-(aminosulfonyl)-4-chloro-2-((2-furanylmethyl)amino)benzoic acid (furosemide, FUR) are described. The methods are based on acid hydrolysis of FUR to free primary aromatic amine and diazotization followed by coupling with N-1-napthylethylene diamine (NEDA) (method A) or 4,5-dihydroxynaphthalene-2,7-disulfonic acid (chromotropic acid, CTA) (method B). The colored reaction product can be measured spectrophotometrically at 520 nm (method A) or 500 nm (method B). Beer’s law is obeyed over the ranges of 1.75–21.0 μg mL⁻¹ and 2.5–30.0 μg mL⁻¹, for method A and method B, respectively. Apparent molar absorptivities and Sandell’s sensitivities (in L mol⁻¹ cm⁻¹ and μg cm⁻² per 0.001 absorbance unit, respectively) were 1.34 × 10⁴ and 0.0253 using NEDA as the coupling agent, and 8.5 × 10³ and 0.0389 using CTA for the same purpose. Analysis of solutions containing seven different concentrations of FUR gave a correlation coefficient of 0.9979 using NEDA and 0.9984 using CTA, while the slope and the correlation coefficient of the regression equation were calculated. The reaction stoichiometry in both methods was evaluated by the limiting logarithmic method and was found to be 1: 1 (diazotized FUR: NEDA or diazotized FUR: CTA). The methods were successfully applied to the determination of FUR in spiked human urine and in pharmaceutical formulations. The recovery of FUR from spiked urine was satisfactory resulting in the values of (109.4 ± 4.37) % using NEDA and (113.0 ± 4.74) % using CTA. Results of the analysis of pharmaceuticals demonstrated that the proposed procedures are at least as accurate and precise as the official method while a statistical analysis indicated that there was no significant difference between the results obtained by the proposed methods and those of the official method.     

Efficient strategy for the selective determination of dopamine in human urine by molecularly imprinted solid‐phase extraction

An efficient molecularly imprinted solid‐phase extraction protocol was developed for the separation of dopamine (DA) from human urine. After successful validation of the analytical method using high‐performance liquid chromatography coupled with fluorescence detection, a new strategy for the selective determination of DA in the presence of norepinephrine and epinephrine in human urine was presented. In the proposed protocol, the LODs and quantification for DA were 166 ± 36 and 500 ± 110 nmol/L, respectively, and the total recoveries of DA in the range of 1–15 μmol/L varied between 98.3 and 101.1%. DA was detected in the real urine samples at the level of 47–167 μg/L (0.250–0.895 μmol/L). The superiority of the novel analytical strategy was shown by comparison with the results obtained for a commercially available imprinted sorbent.     

以OT为底物固体电极伏安法测定辣根过氧化物酶及其标记物的研究

以玻碳电极为工作电极研究了邻联甲苯胺(OT)为底物微分脉冲伏安法测定辣根过氧化物酶(HRP)及其标记物的方法。HRP能够催化H2O2氧化OT,其反应产物在玻碳电极上-0.58V(vs.Ag/AgCl)左右被还原产生一个灵敏的还原峰,还原峰电流随着酶浓度的增大而增大,借助此还原电流可以测定HRP,并进而可用于以HRP为标记物的酶免疫分析。对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究,在最佳实验条件下测定游离HRP的线性范围是2.0×10-9～4.0×10-8g/mL,检出限为1.6×10-9g/mL;测定游离的酶标记物(IgG HRP),稀释范围为1∶2000～1∶400000,最大稀释比为1∶400000。     

Sensitive determination of probenecid in urine samples by reversed-phase liquid chromatography and UV-visible detection using solid-phase extraction techniques for sample clean-up

Summary This study describes a rapid method for the determination of probenecid in human urine by liquid chromatography with UV detection at 254 nm, after clean-up through a C8 solid-phase extraction column. Liquid chromatography was carried out on a C18-bonded phase using an acetonitrile-acetate buffer (pH=4) gradient elution. Ethacrynic acid was used as internal standard. The system has been applied to the determination of probenecid in the 0.10–100.0 g/ml concentration range; the limit of detection was 5 ng/mL.     

Ion chromatographic determination of iodide in urine and serum using a tubular ion-selective electrode based on a homogeneous crystalline membrane

The use of a laboratory-made iodide ion-selective electrode with tubular configuration and based on a crystalline membrane (AgI/Ag₂S) as the detector for ion chromatographic determination of iodide in urine and serum is described. A CIS reversed-phase column was coated withN-cetylpyridinium chloride to prepare a low-exchange-capacity analytical column and with hexadecyltrimethylammonium bromide to prepare a concentrator pre-column. A 2.0 ml min^–1 flow rate of deionized water and 0.1 mol 1^–1 KNO₃ solution was used for the pre-concentration and for the chromatographic separation, respectively. For optimum performance of the detector a background level of iodide was added into the column effluent. A linear relationship (r = 0.9997) between tubular electrode potential (as peak height) and iodide concentration in the range 5–400 g 1^–1 and a detection limit of 1.47 g 1^–1 were obtained. The method shows good reproducibility for both peak height (2.2% RSD) and retention time (1.3% RSD). Recoveries on its application to the samples were 93.0–100.9% for urine and 91.4–106.0% for serum.     

High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urine

A reversed-phase high-performance liquid chromatography assay for mangiferin in rat plasma and urine was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) containing 3% acetic acid at a flow rate of 1 mL/min. Detection was at 257 and 365 nm for mangiferin in plasma and urine, respectively. The limit of quantitation (LOQ) of mangiferin was 0.6 microg/mL in plasma, and 0.48 microg/mL in urine. The standard curve was linear from 0.6 to 24 microg/mL in plasma, and 0.48 to 24 microg/mL in urine, both intra- and inter-day precision of the mangiferin were determined and their RSD did not exceed 10%. The method provides a technique for rapid analysis of mangiferin in rat plasma and urine, which can be used in pharmacokinetic studies.