Solid-phase microextraction combined with gas chromatography and atomic emission detection for the determination of cyclopentadienylmanganese tricarbonyl and (methylcyclopentadienyl)manganese tricarbonyl in soils and seawaters

A method based on solid-phase microextraction with capillary gas chromatography and microwave-induced plasma atomic emission detection (SPME-GC-MIP-AED) for the determination of cyclopentadienylmanganese tricarbonyl (CMT) and its methyl derivative, (methylcyclopentadienyl)manganese tricarbonyl (MMT) in seawaters and soils was optimized and evaluated. The solvent-free preconcentration step was carried out in the headspace (HS) mode. Element-specific detection and quantification was carried out by monitoring the manganese (259 nm) emission line. A matrix effect was found for the soil samples investigated, so that standard additions were required for their quantification. On the other hand seawaters were analyzed by applying direct calibration against aqueous standards. Detection limits of 13 and 15 pg Mn L(-1) were obtained for CMT and MMT, respectively, in seawaters, and 0.62 and 0.65 pg Mn g(-1), respectively, for the soil samples. The method permitted recoveries of 76-113%, depending on the compound and the sample analyzed, confirming the reliability of the procedure and its suitability for routine monitoring purposes.     

Ion chromatographic preconcentration of Cu and Cd from ultra-high-purity water and determination by electrothermal atomic absorption spectrometry

A method based on preconcentration of Cu and Cd from ultra-high-purity water by ion chromatography (IC) and determination by electrothermal atomic absorption spectrometry is described. A small low-capacity ion-exchange concentrator Dionex HPIC-CG5 and mobile phase of 3 mM pyridine-2,6-dicarboxylic acid (PDCA) are used. Water samples are loaded onto the preconcentration column at a flow-rate ranging from 1 to 3.5 ml min(-1). Large sample volumes (up to 200 ml) can be loaded onto the concentrator without losing metal ions. Elution is carried out in the reverse direction of sample loading and the volumes of effluent are as small as 0.150 and 0.200 ml for copper and cadmium, respectively. Under these conditions the preconcentrated ions coelute. The detection limits, based on the Hubaux-Vos method, for Cu using a 1300-fold preconcentration in the IC step was found to be 1 pg ml(-1), and was limited due to impurity in PDCA, while the detection limit found for Cd using a 1000-fold preconcentration was 0.02 pg ml(-1). Ultra-high-purity water produced by a Millipore system is successfully analysed by the proposed method and the content of Cu and Cd are found to lie in the range 1-10 pg ml(-1).     

Determination of steroid sex hormones in urine matrix by stir bar sorptive extraction based on monolithic material and liquid chromatography with diode array detection

A simple, rapid and sensitive method for simultaneous determination of six steroid sex hormones in urine matrix was developed by the combination of stir bar sorptive extraction (SBSE) with high performance liquid chromatography (HPLC) and diode array detection (DAD). A poly (methacrylic acid stearyl ester-ethylene dimethacrylate) was synthesized and selected as SBSE extraction medium. To achieve the optimum extraction performance, several parameters, including agitation speed, desorption solvent, extraction and desorption time, pH value, inorganic salt and organic solvent content of the sample matrix were investigated. Under the optimized experimental conditions, low detection limits (S/N=3) and quantification limits (S/N=10) of the proposed method for the target compounds were achieved within the range of 0.062-0.38 and 0.20-1.20 ng/mL, respectively from spiked urine, respectively. The calibration curves of six steroid sex hormones showed good linearity ranging from 1.0 to 200 ng/mL with linear coefficient R2 values above 0.990. Good method reproducibilities presented as intra- and inter-day precisions were also found with the R.S.D.s less than 9.2 and 10.0%, respectively. Finally, the proposed method was successfully applied to the determination of the target compounds in a urine sample from a pregnant woman.     

Quantitative determination of nicotinic acid in micro liter volume of urine sample by drop-to-drop solvent microextraction coupled to matrix assisted laser desorption/ionization mass spectrometry

Drop-to-drop solvent microextraction (DDSME) coupled with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for quantitative determination of nicotinic acid in one drop of urine sample has been proposed. All parameters, such as type of organic solvent, extraction time, exposure volume solvent, pH of the sample solution that affecting the separation and preconcentration of nicotinic acid were investigated. Under the optimal conditions, the detection limit of the method was 20 ng mL(-1) and the relative standard deviations (RSD) for determination of the nicotinic acid were in the range of 8.0-12.5%. The calculated calibration curves gave linearity in the range of 80-1000 ng mL(-1). The main advantages of the proposed method are simple, fast, and small amount of sample solution is used for separation and preconcentration of nicotinic acid. This method could be also useful for the analysis of other interested analytes in small volume of biological samples, like plasma, saliva and urine, where the availability of samples are limited.     

Flow-injection flame atomic absorption spectrometric determination of trace amounts of cadmium in solid and semisolid milk products coupling a continuous ultrasound-assisted extraction system with the online preconcentration on a chelating aminomethylphosphoric acid resin

A new sensitive and low-cost method that combines continuous acid extraction, online preconcentration, and flame atomic absorption spectrometry for cadmium determination at microg levels in solid and semisolid milk products is described. A continuous ultrasound-assisted extraction system is used to carry out the dynamic acid extraction step. The acid extract is preconcentrated online on a minicolumn packed with a chelating resin (Chelite P, with aminomethylphosphoric acid groups), and the retained cadmium is eluted with hydrochloric acid and continuously monitored by flame atomic absorption spectrometry. An experimental design (Plackett-Burman 2-6*3/16) is used to optimize the continuous extraction and the preconcentration step. The method allowed a total sampling frequency of 28 samples/h. A good precision of the whole procedure (3.0% relative standard deviation) for a cheese sample containing 0.103 +/- 0.004 microg/g Cd (dry mass), a high enrichment factor (20.5), and detection and quantification limits of 0.014 and 0.067 microg/g, respectively, for a 60 mg of sample were obtained with this methodology. The method was successfully applied to the determination of trace amounts of cadmium in solid and semisolid milk products, such as cheese and yogurt samples.     

Optimization of a clean-up procedure for the determination of heterocyclic aromatic amines in urine by field-amplified sample injection-capillary electrophoresis-mass spectrometry

Heterocyclic amines (HAs), generated when proteinaceous food is cooked, are of special interest since they can be carcinogenic for humans. In this paper, the optimization of a clean-up procedure for the isolation and preconcentration of 15 heterocyclic amines in urine samples is described. The method proposed combines liquid extraction on a solid support of diatomaceous earth with solid-phase extraction in cartridges. Tests were performed on several cartridges containing graphitic carbon or mixed phases, i.e., combining reversed-phase and cation-exchange mechanism, and the best results were obtained with Oasis MCX. The optimized purification method was applied to the quantification of heterocyclic amines in hydrolyzed spiked human urine. The method was carried out by capillary electrophoresis (CE) coupled to mass spectrometry (MS) and applying field-amplified sample injection (FASI) as in-line preconcentration procedure. We obtained detection limits down to 0.3 ng/ml of urine and errors lower than 17%.     

Enantioselective column coupled electrophoresis employing large bore capillaries hyphenated with tandem mass spectrometry for ultra‐trace determination of chiral compounds in complex real samples

A new multidimensional analytical approach for the ultra‐trace determination of target chiral compounds in unpretreated complex real samples was developed in this work. The proposed analytical system provided high orthogonality due to on‐line combination of three different methods (separation mechanisms), i.e. (1) isotachophoresis (ITP), (2) chiral capillary zone electrophoresis (chiral CZE), and (3) triple quadrupole mass spectrometry (QqQ MS). The ITP step, performed in a large bore capillary (800 μm), was utilized for the effective sample pretreatment (preconcentration and matrix clean‐up) in a large injection volume (1–10 μL) enabling to obtain as low as ca. 80 pg/mL limits of detection for the target enantiomers in urine matrices. In the chiral CZE step, the different chiral selectors (neutral, ionizable, and permanently charged cyclodextrins) and buffer systems were tested in terms of enantioselectivity and influence on the MS detection response. The performance parameters of the optimized ITP – chiral CZE‐QqQ MS method were evaluated according to the FDA guidance for bioanalytical method validation. Successful validation and application (enantioselective monitoring of renally eliminated pheniramine and its metabolite in human urine) highlighted great potential of this chiral approach in advanced enantioselective biomedical applications.     

Determination of trace levels of benzodiazepine in urine using capillary electrochromatography-time of flight mass spectrometry

This paper details a method for the separation and determination of ten benzodiazepines in urine using CEC-MS(TOF) and an hexyl acrylate-based porous monolith. The TOF mass spectrometer provides an exact mass of protonated benzodiazepines to three decimal places (1-6 ppm). This high selectivity along with the CEC separation, provides an effective method for the identification of benzodiazepines. Linearity is satisfactory for all compounds in the concentration range of 25-500 ng/mL for lorazepam and 12.5-500 ng/mL for the others. The RSDs are between 1.4-2.3% for retention times and 1.1-9.2% for relative areas. Using the monolithic stationary phase, a preconcentration step is achievable and permits an 75-140-fold improvement in sensitivity. This strategy permits the quantification of these drugs down to 1 ng/mL in urine. This method was used for the analysis of benzodiazepines in spiked urine samples.     

Monitoring of 6-chloronicotinic acid in human urine by gas chromatography-tandem mass spectrometry as indicator of exposure to the pesticide imidacloprid

A new analytical method for determining 6-chloronicotinic acid (6-ClNA) in human urine is proposed. 6-ClNA is the main metabolite in warm-blooded animals after exposure to the insecticide imidachloprid. 6-ClNA was extracted from human urine using solid phase extraction (SPE) with laboratory-made cartridges of Amberlite XAD-4. A clean-up step and a derivatization process were carried out prior to gas chromatography-tandem mass spectrometric (GC-MS-MS) determination. A study on the influence of pH in the extraction process revealed that it affects the analyte extraction efficiency. A working pH zone was defined between 0.8 and 2.8. Calibration curves were studied in the concentration range of 0.5-100 ng mL(-1) and showed good linearity. Limits of detection and determination of the method were 16 and 56 pg mL(-1) respectively. The mean recovery at 10 and 100 ng mL(-1) was between 97.2 and 102.1% and the repeatability was lower than 5.4% in all cases. The analysis of urine samples of five agricultural workers from Almería (Spain) did not detect the metabolite.     

A critical evaluation of the graphite furnace conditions for the direct determination of chromium in urine

A critical study of the conditions for the direct determination of Cr in urine using GFAAS was carried out. A chemometric approach showed that the most important parameters that influence the efficiency of atomic Cr cloud formation were the atomization from the tube wall and the proper implementation of a pyrolysis step. The established procedure does not require any sample pre-treatment thus minimizing the risk of contamination. The Cr content in urine was determined using a calibration curve prepared with Cr spiked urine, easily correcting all potential matrix interferences. The heating program proposed took 68 s for a 30 μL urine sample. The procedure was applied for the determination of Cr in urine to investigate the absorption rate of Cr picolinate. The limit of detection and the characteristic mass for a 30 μL urine sample were 0.18 μg/L and 5.4 pg, respectively.     

Ionic liquid-based dynamic liquid-phase microextraction: application to the determination of anti-inflammatory drugs in urine samples

Dynamic liquid-phase microextraction (dLPME) using an ionic liquid as acceptor phase is proposed for the determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in human urine samples for the first time. The extraction is carried out in a simple and automatic flow configuration. The chemical affinity between the extractant (1-butyl-3-methylimidazolium hexafluorophosphate) and the analytes permits a selective isolation of the drugs from the sample matrix allowing also their preconcentration. The whole analytical method has been optimized taking into account all the chemical, physical and hydrodynamic variables. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.1-10 microg mL(-1), allowing their determination at therapeutic and toxic levels. Limits of detection were in the range from 38 ng mL(-1) (indomethacin) to 70 ng mL(-1) (naproxen). The repeatability of the proposed method expressed as RSD (n=5) varied between 2.1% (flurbiprofen) and 3.8% (tolmetin).     

Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.     

Quantification of clenbuterol in equine plasma,urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry

Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method involved liquid-liquid extraction (LLE), separation by liquid chromatography (LC) on a short cyano column, and pseudo multiple reaction monitoring (pseudo-MRM) by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS). At very low concentrations (picograms of CBL/mL), LLE produced better extraction efficiency and calibration curves than solid-phase extraction (SPE). The operating parameters for electrospray QTOF and yield of the product ion in MRM were optimized to enhance sensitivity for the detection and quantification of CBL. The quantification range of the method was 0.013-10 ng of CBL/mL plasma, 0.05-20 ng/0.1 mL of urine, and 0.025-10 ng/g tissue. The detection limit of the method was 13 pg/mL of plasma, 50 pg/0.1 mL of urine, and 25 pg/g of tissue. The method was successfully applied to the analysis of CBL in plasma, urine and various tissue samples, and in pharmacokinetic (PK) studies of CBL in the horse. CBL was quantified for 96 h in plasma and 288 h in urine post-administration of CLB (1.6 micro g/kg, 2 x daily x 7 days). This method is useful for the detection and quantification of very low concentrations of CBL in urine, plasma and tissue samples.     

Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography-mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-beta-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo-N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE-LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE-LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-deethylzaleplon.     

Solid phase extraction using a sulfoxide adsorbent for preconcentration and separation of Hg(II) in natural water followed by ICP-MS measurements

A separation and preconcentration method based on solid-phase extraction using sulfoxide adsorbent was developed for the determination of Hg(II) in natural water samples by inductively coupled plasma mass spectrometry (ICP-MS). The sulfoxide adsorbent was packed into a commercially available syringe-driven column (with a bed volume of 1.0 mL), which permitted off-line sample loading and on-line elution/measurement. The optimized operating conditions were as follows: sample condition for Hg(II) adsorption, 0.5% HCl; sample-loading flow rate, 10 mL min(-1); eluent for recovering Hg(II), 1% cysteine water solution. A test using multi-element mixed solution showed that most trace elements in natural water, except for Bi, could be completely separated from Hg(II). The recoveries of Hg(II) were 99.0 ± 3.2 and 100.7 ± 4.3%, respectively, when 0.64 and 0.16 ng mL(-1) of Hg(II) were added into the test sample. The detection limit of Hg(II) using a quadrupole ICP-MS after 10-fold preconcentration was 1.5 pg mL(-1). The blank value was 2.8 ± 0.5 pg mL(-1).     

Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry

Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable. Copyright (c) 2008 John Wiley & Sons, Ltd.     

A critical evaluation of the graphite furnace conditions for the direct determination of chromium in urine

A critical study of the conditions for the direct determination of Cr in urine using GFAAS was carried out. A chemometric approach showed that the most important parameters that influence the efficiency of atomic Cr cloud formation were the atomization from the tube wall and the proper implementation of a pyrolysis step. The established procedure does not require any sample pre-treatment thus minimizing the risk of contamination. The Cr content in urine was determined using a calibration curve prepared with Cr spiked urine, easily correcting all potential matrix interferences. The heating program proposed took 68 s for a 30 μL urine sample. The procedure was applied for the determination of Cr in urine to investigate the absorption rate of Cr picolinate. The limit of detection and the characteristic mass for a 30 μL urine sample were 0.18 μg/L and 5.4 pg, respectively. Received: 22 June 1998 / Revised: 13 January 1999 / Accepted: 18 January 1999     

Polyaniline-silica doped with oxalic acid as a novel extractor phase in thin film solid-phase microextraction for determination of hormones in urine

In this study, different polyanilines were synthesized and evaluated for the determination of three hormones, including 17-β-estradiol, 17-α-ethinylestradiol, and estrone, in urine using a novel methodology based on thin film solid-phase microextraction technique, employing the sampling well plate system. The extractor phases, designated as polyaniline doped with hydrochloric acid, polyaniline doped with oxalic acid, polyaniline-silica doped with hydrochloric acid, and polyaniline-silica doped with oxalic acid, were characterized by electrical conductivity measurements, scanning electron microscopy, and Fourier transform infrared spectroscopy. The optimized extraction conditions were composed of 1.5 mL of urine and pH adjusted to 10, with no need to dilute sample and the desorption step, 300 μL of acetonitrile was used. The calibration curves were performed in the sample matrix, with detection and quantification limits ranged from 0.30 to 3.03 μg L⁻¹ and from 1.0 to 10.0 μg L⁻¹, respectively, with r ≥ 0.9969. The relative recoveries ranged from 71% to 115%, and intraday precision showed values ≤12% and interday ≤20%. The applicability of the method was successfully evaluated, and six urine samples from female volunteers were analyzed. The analytes were not detected or were below the limits of quantification in these samples.     

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies.

Determination of tetracyclines in human urine samples by capillary electrophoresis in combination with field amplified sample injection

A sensitive method using CZE‐UV detection has been developed for the determination of five tetracycline antibiotics in human urine samples. To improve the sensitivity of the method, an on‐line preconcentration strategy, named field‐amplified sample injection, has been developed, based on the electrokinetic injection of the sample, which requires only a 1:100 dilution with sample solvent before injection. Under optimum conditions, sensitivity enhancement factors ranged from 450 to 800 for the studied compounds. The applicability of the proposed method was demonstrated by the determination of these antibiotics in spiked urine samples. The limits of quantification were lower than 0.8 mg/L and the precision (intra‐ and inter‐day), expressed as %RSD was below 14%. Recoveries ranged from 92.1 to 96.7%. Thus, the proposed procedure is a simple, fast and efficient strategy which could be used as therapeutic drug monitoring in human urine samples.