Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification

A competitive microplate fluoroimmunoassay was developed for the determination of human serum albumin in urine. It is based on the use of biotinylated CdTe quantum dots (QDs) whose synthesis is optimised in terms of storage stability, purification, and signal-to-noise ratio. The bioconjugated QDs were characterised by gel chromatography and gel electrophoresis. Storage stability and quantum yield were investigated. The excitation/emission wavelengths are 360/620?nm. The immunoassay of human serum albumin in urine has a working range from 1.7 to 10?μg.mL^?1, and the limit of detection is 1.0?μg.mL^?1.

Electrochemically controlled solid-phase micro-extraction of proline using a nanostructured film of polypyrrole, and its determination by ion mobility spectrometry

We report on a fast, simple and accurate method for the determination of proline in urine samples by employing a nanostructured film of conducting polypyrrole for electrochemically controlled solid-phase microextraction, and ion mobility spectrometry (IMS) for detection. This method has the advantages of simple sample preparation and a sensitivity of IMS to proline that is higher than that for other amino acids. The calibration curve is linear in the range of 0.5–60 μg L^?1 (4–521 nmol L^?1), and the detection limit is 0.2 μg L^?1. The electrochemical potentials for uptake and release were optimized. The method was successfully applied to the clean-up and quantitation of trace amounts of proline in urine samples.

Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography–tandem mass spectrometry

The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing–polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C₁₈ column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was ⁹⁰TLQLHVDKAVSGLRSLTTLLRALGAQKE¹¹⁷ (V₁₁). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa (n?=?3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing.

A novel reversed-phase HPLC method for the determination of urinary creatinine by pre-column derivatization with ethyl chloroformate: comparative studies with the standard Jaffé and isotope-dilution mass spectrometric assays

Creatinine is an important biomarker for renal function diagnosis and normalizing variations in urinary drug/metabolites concentration. Quantification of creatinine in biological fluids such as urine and plasma is important for clinical diagnosis as well as in biomonitoring programs and urinary metabolomics/metabonomics research. Current methods for creatinine determination either are nonselective or involve the use of expensive mass spectrometers. In this paper, a novel reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of creatinine of high hydrophilicity by pre-column derivatization with ethyl chloroformate is presented. N-Ethyloxycarbonylation of creatinine significantly enhanced the hydrophobicity of creatinine, facilitating its chromatographic retention as well as quantification by HPLC. Factors governing the derivatization reaction were studied and optimized. The developed method was validated and applied for the determination of creatinine in rat urine samples. Comparative studies with isotope-dilution mass spectrometric method revealed that the two methods do not yield systematic differences in creatinine concentrations, indicating the HPLC method is suitable for the determination of creatinine in urine samples.

Application of UPLC-QTOF-MS in MSE mode for the rapid and precise identification of alkaloids in goldenseal (Hydrastis canadensis)

Here, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS^E mode (UPLC-QTOF-MS^E) for the sensitive, fast, and effective characterization of alkaloids in goldenseal (Hydrastis canadensis). This approach allowed identification of alkaloids using a cyclic low and high collision energy spectral acquisition mode providing simultaneous accurate precursor and fragment ion mass information. A total of 45 compounds were separated and 40 of them characterized including one new compound and 7 identified for the first time in goldenseal. The spectral data obtained using this method is comparable to those obtained by conventional LC-MSⁿ. However, the UPLC-QTOF-MS^E method offers high chromatographic resolution with structural characterization facilitated by accurate mass measurement in both MS and MS/MS modes in a single analytical run; this makes it suitable for the rapid analysis and screening of alkaloids in plant extracts.

Analysis of dextromethorphan and pseudoephedrine in human plasma and urine samples using hollow fiber-based liquid–liquid–liquid microextraction and corona discharge ion mobility spectrometry

We report on the use of hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) followed by corona discharge ion mobility spectrometry for the determination of dextromethorphan and pseudoephedrine in urine and plasma samples. The effects of pH of the donor phase, stirring rate, ionic strength and extraction time on HF-LLLME were optimized. Under the optimized conditions, the linear range of the calibration curves for dextromethorphan in plasma and urine, respectively, are from 1.5 to 150 and from 1 to 100 ng mL^?1. The ranges for pseudoephedrine, in turn, are from 30 to 300 and from 20 to 200 ng mL^?1. Correlation coefficients are better than 0.9903. The limits of detection are 0.6 and 0.3 ng mL^?1 for dextromethorphan, and 8.6 and 4.2 ng mL^?1 for pseudoephedrine in plasma and urine samples, respectively. The relative standard deviations range from 6 to 8%.

Comparison of the Mahalanobis Distance and Pearson’s χ2 Statistic as Measures of Similarity of Isotope Patterns

To extract a genuine peptide signal from a mass spectrum, an observed series of peaks at a particular mass can be compared with the isotope distribution expected for a peptide of that mass. To decide whether the observed series of peaks is similar to the isotope distribution, a similarity measure is needed. In this short communication, we investigate whether the Mahalanobis distance could be an alternative measure for the commonly employed Pearson’s χ² statistic. We evaluate the performance of the two measures by using a controlled MALDI-TOF experiment. The results indicate that Pearson’s χ² statistic has better discriminatory performance than the Mahalanobis distance and is a more robust measure.

Determination of N-acetylcysteine via its effect on the aggregation of gold nanoparticles

The effect of thiol compounds on the kinetics of the aggregation of gold nanoparticles in the presence of the cationic surfactant cetyltrimethyl ammonium bromide has been studied. It was applied to the determination of N-acetylcysteine using the stopped-flow mixing technique along with light scattering detection. The signal obtained was measured after about 5?s, and gave the analytical information for a calibration graph in the concentration range from 2.9 to 60???mol?L^?1 of N-acetylcysteine, and a detection limit of 0.87???mol?L^?1. The effect of other thiols on the system is also described. The relative standard deviation ranges between 0.6% and 3.5%. The method was applied to the determination of N-acetylcysteine in several pharmaceutical samples with recoveries that range from 97.7% to 101.1%.

A colorimetric method for the determination of lead(II) ions using gold nanoparticles and a guanine-rich oligonucleotide

We have developed a colorimetric method for the determination of Pb(II) ions. It is based on the use of gold nanoparticles and a guanine-rich synthetic oligonucleotide. On addition of Pb(II), the color of the solution turns from red to blue. The ratio of the UV-vis absorption at 630?nm and 525?nm is proportional to the concentration of Pb(II) ions in the range from 10 to 100?nM, and the detection limit is 20?nM. Other metal ions do not interfere if present in up to a 10-fold molar excess. The method was successfully applied to the detection of Pb(II) in lake water and urine. The recovery in case of spiked samples is 92%. The results show that this method is sensitive, simple and fast.

Analysis of narcotic drugs in biological samples using hollow fiber liquid�Cphase microextraction and gas chromatography with nitrogen phosphorus detection

We report on a method for trace analysis of the narcotic drugs alfentanil, fentanyl, and sufentanil in plasma and urine. Two?Cphase hollow fiber liquid?Cphase microextraction was combined with GC using nitrogen?Cphosphorus detection. Experimental parameters were optimized to give a viable analytical procedure whose limits of detection range from 8 to 15?ng L^?1 (at an S/N of 3). The calibration curves are linear between 0.1 and 50???g L^?1, with squared correlation coefficients (r ²) between 0.9953 and 0.9979. Precision values range from 2.4% to 3.3% (intra?Cday RSD) and 3.2 to 6.3% (inter?Cday RSD). The relative recoveries varied from 27.8% to 84.6% (for spiked plasma) and 75 to 85.2% (for spiked urine). The method consumes little solvent, is simple, fast, inexpensive, and well suitable for the analysis of complicated matrices.

Micro-solid phase extraction of ochratoxin A, and its determination in urine using capillary electrophoresis

We describe a simple, environmentally friendly and selective technique for the determination of ochratoxin A (OTA) in urine. It involves (a) the use of a molecularly imprinted polymer as a sorbent in micro-solid-phase extraction in which the sorbent is contained in a propylene membrane envelope, and (b) separation and detection by capillary electrophoresis (CE). Under optimized conditions, response is linear in the range between 50 and 300 ng mL^?1 (with a correlation coefficient of 0.9989), relative standard deviations range from 4 to 8 %, the detection limit for OTA in urine is 11.2 ng mL^?1 (with a quantification limits of 32.5 ng mL^?1) which is lower than those of previously reported methods for solid-phase extraction combined with CE. The recoveries of OTA from urine spiked at levels of 50, 150 and 300 ng mL^?1 ranged from 93 to 97 %.

Cross-validation and evaluation of the performance of methods for the elemental analysis of forensic glass by μ-XRF, ICP-MS, and LA-ICP-MS

Elemental analysis of glass was conducted by 16 forensic science laboratories, providing a direct comparison between three analytical methods [micro-x-ray fluorescence spectroscopy (μ-XRF), solution analysis using inductively coupled plasma mass spectrometry (ICP-MS), and laser ablation inductively coupled plasma mass spectrometry]. Interlaboratory studies using glass standard reference materials and other glass samples were designed to (a) evaluate the analytical performance between different laboratories using the same method, (b) evaluate the analytical performance of the different methods, (c) evaluate the capabilities of the methods to correctly associate glass that originated from the same source and to correctly discriminate glass samples that do not share the same source, and (d) standardize the methods of analysis and interpretation of results. Reference materials NIST 612, NIST 1831, FGS 1, and FGS 2 were employed to cross-validate these sensitive techniques and to optimize and standardize the analytical protocols. The resulting figures of merit for the ICP-MS methods include repeatability better than 5 % RSD, reproducibility between laboratories better than 10 % RSD, bias better than 10 %, and limits of detection between 0.03 and 9 μg g^?1 for the majority of the elements monitored. The figures of merit for the μ-XRF methods include repeatability better than 11 % RSD, reproducibility between laboratories after normalization of the data better than 16 % RSD, and limits of detection between 5.8 and 7,400 μg g^?1. The results from this study also compare the analytical performance of different forensic science laboratories conducting elemental analysis of glass evidence fragments using the three analytical methods.

Selection and identification of ssDNA aptamers recognizing zearalenone

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z₃₁, with a dissociation constant (K _d) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10^?9 to 3.14?×?10^?5 M for ZEN, and the detection limit was 7.85?×?10^?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

Heavy element stable isotope ratios : analytical approaches and applications

Continuous developments in inorganic mass spectrometry techniques, including a combination of an inductively coupled plasma ion source and a magnetic sector-based mass spectrometer equipped with a multiple-collector array, have revolutionized the precision of isotope ratio measurements, and applications of inorganic mass spectrometry for biochemistry, geochemistry, and marine chemistry are beginning to appear on the horizon. Series of pioneering studies have revealed that natural stable isotope fractionations of many elements heavier than S (e.g., Fe, Cu, Zn, Sr, Ce, Nd, Mo, Cd, W, Tl, and U) are common on Earth, and it had been widely recognized that most physicochemical reactions or biochemical processes induce mass-dependent isotope fractionation. The variations in isotope ratios of the heavy elements can provide new insights into past and present biochemical and geochemical processes. To achieve this, the analytical community is actively solving problems such as spectral interference, mass discrimination drift, chemical separation and purification, and reduction of the contamination of analytes. This article describes data calibration and standardization protocols to allow interlaboratory comparisons or to maintain traceability of data, and basic principles of isotope fractionation in nature, together with high-selectivity and high-yield chemical separation and purification techniques for stable isotope studies.

Preconcentration of ultra-trace amounts of iron and antimony using ion pair solid phase extraction with modified multi-walled carbon nanotubes

Ion pair solid phase extraction was applied to the simultaneous preconcentration of iron and antimony. The ion pairs consisting of FeCl₄ ^? or SbCl₄ ^? anions and the benzyldimethyltetradecyl ammonium cation were formed on the surface of multi-walled carbon nanotubes, then eluted with nitric acid, and the elements finally quantified by ETAAS. The adsorption capacities of the impregnated MWCNTs are 9.2 mg g^?1 for iron and 27.5 mg g^?1 for antimony. The following analytical figures of merit were determined for iron and antimony, respectively: Enrichment factors of 210 and 230, assay precisions of ±5.3 % and ±4.8 %, linear calibration plots from 0.7 to 9.4 and 13.0 to 190 ng L^?1, and detection limits of 0.17 and 3.5 ng L^?1. The method was applied to the determination of iron and antimony in human hair, synthetic sample, and to the certified reference materials gold ore (MA-1b) and trace elements in water (SRM 1643d).

Sensitive detection of trace hemoglobin using fluorescence method based on functionalized quantum dots

The fluorescence quenching of quantum dots by hemoglobin has been demonstrated to depend on surface functionalization, and this property has been utilized to construct a novel fluorescent method for rapid, sensitive, and selective detection of trace hemoglobin in urine at microgram level. This method shows low interference and high selectivity for hemoglobin with a limit of detection of 4.3 μg L^?1 in water and 66.1 μg L^?1 in urine, which are lower than those of currently used methods in labs and clinics. Spike and recovery tests in raw, acidified, and alkalized urine samples exhibit good recovery rates for the spiked concentrations close to the limit of detection.

Rapid and sensitive HILIC–MS/MS analysis of carnitine and acetylcarnitine in biological fluids

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography–tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

Centri-voltammetry for biosensing systems: biocentri-voltammetric xanthine detection

Centri-voltammetry is a method that combines centrifugation and voltammetry. This method, developed by our group in 2003, yielded promising results when applied to trace analysis of metal ios. We demonstrate here the first application of centri-voltammetry to biosensing systems. A xanthine biosensor was constructed by immobilizing xanthine oxidase on a planar platinum electrode which then was placed at the bottom of a centri-voltammetric cell. The experimental parameters were optimized to give two linear ranges. The first is from 0.1 to 1???M, and the second from 5 to 50???M. The RSD is 3.4 (n?=?5). The biosensor was applied to the determination of xanthine in wine and in urine. Calculated recoveries are 101?±?0.61% (n?=?3) for wine samples, and 102?±?0.556% (n?=?3) for urine samples.

Determination of cocaine and methadone in urine samples by thin-film solid-phase microextraction and direct analysis in real time (DART) coupled with tandem mass spectrometry

The use of thin-film solid-phase microextraction (SPME) as the sampling preparation step before direct analysis in real time (DART) was evaluated for the determination of two prohibited doping substances, cocaine and methadone, in urine samples. Results showed that thin-film SPME improves the detectability of these compounds: signal-to-blank ratios of 5 (cocaine) and 13 (methadone) were obtained in the analysis of 0.5 ng/ml in human urine. Thin-film SPME also provides efficient sample cleanup, avoiding contamination of the ion source by salt residues from the urine samples. Extraction time was established in 10 min, thus providing relatively short analysis time and high throughput when combined with a 96-well shaker and coupled with DART technique.