Analysis of carbamazepine and its five metabolites in serum by large-volume sample stacking–sweeping capillary electrophoresis

This study establishes a method, using different buffer conductivities and large-volume sample stacking (LVSS)–sweeping capillary electrophoresis, for analysis of carbamazepine (CBZ) and its five metabolites in serum. The capillary (50/60 cm) was filled with a high concentration of background electrolyte (150 mM phosphate, pH?3.5, containing 15 % methanol), followed by a large volume of samples (10 psi, 20 s) with low-concentration buffers (5 mM phosphate, pH?3.5, with 5 % methanol). When high voltage was applied (?20 kV), the sodium dodecyl sulfate (SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes decelerated at the boundary between low- and high-conductivity buffers. Finally, a narrow sample zone was formed. The procedure of sweeping and separation was simultaneously carried out by a sweeping buffer (150 mM phosphate, pH?3.5) with 15 % methanol and 50 mM SDS added, and the detection was performed by UV at 214 nm. The method was validated for linearity (r?≧?0.997), precision, and accuracy. The calibration curves were established for CBZ and its five metabolites between 0.03–25 and 0.03–3 μg/mL. The limits of detection (S/N?=?3) were 0.01 μg/mL for each analyte. Compared with simple MEKC (0.5 psi, 5 s), this system can improve the sensitivity about 300-fold. Finally, this method was successfully applied to five patients, who had taken 200 mg CBZ daily, and CBZ levels were found to be from 3.72 to 5.82 μg/mL.

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.     

Simultaneous Determination of Anabolic Androgenic Steroids and Their Esters in Hair by LC–MS–MS

A liquid chromatographic-tandem mass spectrometric method for the simultaneous determination of anabolic androgenic steroids and their esters in hair has been developed. The hair sample was treated with methanol to extract the esters, followed by alkaline digestion for optimum recovery of the anabolic androgenic steroids. After liquid–liquid extractions, the extract was dried, redissolved and analyzed by multiple reaction monitoring with a quadrupole mass spectrometer. The lower limits of detection ranged from 0.001 to 0.020 ng mg^?1 for the 21 analytes. The applicability of the method was demonstrated using guinea pig hair samples gained from controlled experiments.     

Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography-high resolution mass spectrometry

Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled “not for human consumption” and considered “legal highs”. Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3’-substituted, N-alkyl-substituted, ring-substituted, methylenedioxy-substituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 μL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5–1 to 100 μg/L, with limits of detection of 0.25–1 μg/L. Imprecision (n?=?20) was <15.9 % and accuracy (n?=?20) 85.2–118.1 %. Extraction efficiency was 78.9–116.7 % (CV 1.4–16.7 %, n?=?5), process efficiency 57.7–104.9 %, and matrix effects from ?29.5 % to 1.5 % (CV 1.9–13.1 %, n?=?10). Most synthetic cathinones were stable at 4 °C for 72 h (n?=?27) and after 3 freeze-thaw cycles (n?=?26), but many (n?=?19) were not stable at room temperature for 24 h (losses up to ?67.6 %). The method was applied to authentic urine specimens from synthetic cathinone users. This method provides a comprehensive confirmation method for 28 synthetic cathinones in urine, with good selectivity and specificity.     

A novel stacking method of repetitive large volume sample injection and sweeping MEKC for determination of androgenic steroids in urine

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254 nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10 psi for 20 s, and then stacked at −10 kV for 1 min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at −20 kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r ≧ 0.997) over a range of 5-200 ng mL⁻¹ for T, 20-200 ng mL⁻¹ for E and 0.5-500 ng mL⁻¹ for EG. The limits of detection were 1.0 ng mL⁻¹ for T, 5.0 ng mL⁻¹ for E and 200.0 pg mL⁻¹ for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n = 3) and inter-day (n = 5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (∼2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes’ urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen.     

Direct Determination of Barbiturates in Urine by Capillary Electrophoresis Using a Capillary Coated Dynamically with Polycationic Polymers

A new, simple and rapid capillary electrophoresis method, using hexadimethrine bromide (HDB) as electro-osmotic flow modifier, has been developed for the identification and determination of nine barbiturates, barbital acid, barbital, phenobarbital, pentobarbital, amobarbital, thiobarbituric acid, butobarbital, N-methyl-5-phenyl-ethyl barbital acid and 5-cyclohexenyl-5-ethyl barbital acid in urine with UV detection at 200 nm. The applied voltage was ?25 kV and the capillary temperature was kept constant at 25 °C. The effects of buffer pH, the concentration of HDB and the concentration of α-cyclodextrin were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.04% (w/v) HDB and 2.06 mM α-cyclodextrin. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. The correlation coefficients were from 0.9990 to 0.9997. The relative standard deviations of migration times and peak areas were <3.84 and 5.45% (intra-day). The nine barbiturates in urine were successfully determined within 7 min, without a prior preparation step and the method is useful for the investigation of intoxication.     

Sensitive Analysis of 5-(4,6-Dichloro-s-triazin-2-ylamino)fluorescein-Labeled Catecholamines by Mixed MEKC–LIF

Mixed micellar electrokinetic chromatography with laser-induced fluorescence detection has been used for analysis of the catecholamines norepinephrine, epinephrine, and dopamine. The fluorescent reagent 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein was used to label the three compounds. The reaction rate increased with increasing alcohol concentration in the derivatization buffer. Under the optimum conditions the derivatization reaction was complete within 10 min. The separation was performed with 40 mM sodium cholate, 30 mM sodium dodecyl sulfate, 30 mM sodium borate (pH 9.6), and acetonitrile 8.0% (v/v) as running buffer. The applied potential was 25 kV and the capillary temperature was 25 °C. The detection limits for norepinephrine, epinephrine, and dopamine were 3.3, 0.25, and 1.26 nM. The method was successfully applied to monitoring of these catecholamines in human urine. Recovery of the three analytes ranged from 93.2 to 105.8%.     

Analysis of methotrexate and its eight metabolites in cerebrospinal fluid by solid-phase extraction and triple-stacking capillary electrophoresis

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5–7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu) _{n, n = 2–5}), 0.2 μM for MTX-(Glu)₆, and 0.3 μM for 2,4-diamino-N ¹⁰-methylpteroic acid (DAMPA) and MTX-(Glu)₇. Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.     

Separation and stacking of iodine species from seafood using surfactant-coated multiwalled carbon nanotubes as a pseudo-stationary phase in capillary electrophoresis

This article describes an on-line technique for hydrodynamic injections of long sample plugs with simultaneous stacking of iodine species in capillary electrophoresis (CE) with diode array detection. Surfactant-coated multi-walled carbon nanotubes (SC-MWNTs) were used as a pseudostationary phase for the separation of iodate, tetraiodothyronine,triiodothyronine, diiodothyronine, and diiodotyrosine. The effects of MWNTs, concentration of SC-MWNTs and salt, of buffer pH value, injection times were examined. Under the optimized conditions, i.e. detection at 230 nm, a separation voltage 25 kV, a borate running buffer of pH 7.5 and a SC-MWNT concentration of 9 μg?·?mL^?1, the method gave relative standard deviations of the retention times and peak areas in intra-day assays (for n?=?6) and for inter-day assays (for n?=?3) of less than 4.49 and 5.80 %, respectively. The CE method was then applied to the analysis of the above iodine species in (spiked) kelp (kunbu) and porphyra, and recoveries ranged from 81.6 to 98.4 % with RSDs (n?=?3) for extraction repeatability of <3.39 % in all cases.

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Graphical Abstract Surfactant coated multi-walled carbon nanotubes were used as a pseudostationary phase to improve separation in iodine speciation. A highly sensitive stacking method was developed to enhance the detection sensitivity of iodine species. The CE method was then applied to the analysis of iodine species in kelp and porphyra.

     

A new mixed micellar electrokinetic chromatography method for analysis of natural and synthetic anabolic steroids

A simple, rapid and low-costing new mixed surfactant MEKC method has been developed for the analysis of five neutral anabolic steroids in this paper. It was found that the bile salt coupling with Triton X-100 was a suitable bi-micellar surfactant for the separation of these anabolic steroids with similar structure. The separation conditions were optimized in detail. The five natural and synthetic anabolic steroids, such as androstenedione (AD), 19-norandrostenedione (NAD), 1,4-androstadiene-3,17-dione (ADD), methandrostenolone (MA) and methyltestosterone (MT) were separated and detected in an alkaline buffer system (pH 9.0) containing 15 mM Britton-Robinson (BR) buffer, 50 mM sodium cholate (SC) and 0.1% (v/v) Triton X-100 with detection wavelength at 241 nm and 18 kV of separation voltage. Under the optimal conditions, five coexistence neutral steroids were completely separated within 12 min with the detection limits ranged from 0.20 to 0.51 μg/mL. This method was successfully used for detection and confirmation of the anabolic steroid methandrostenolone in methandrostenolone tablets and in the real human urine, GC-MS method was applied to confirm the free methandrostenolone existence in the urine sample in order to validate the reliability of MEKC method.     

Partial filling micellar electrokinetic chromatography analysis of androgens and testosterone derivatives using two sequential pseudostationary phases

Separation of anabolic and androgenic steroids by micellar electrokinetic chromatography (MEKC) has been little studied. Simultaneous separation of the endogenous alpha-epimers testosterone and epitestosterone has not been achieved with any electroseparation technique. Here, a partial filling micellar electrokinetic chromatographic (PF-MEKC) method is described for the analysis of three endogenous steroid hormones (androstenedione, testosterone, epitestosterone) and two synthetic anabolic steroids (fluoxymesterone, methyltestosterone). The resolution efficiency of single-isomer sulphated gamma-cyclodextrins and the surfactants sodium dodecyl sulphate and sodium taurocholate was exploited. The method is based on the sequential introduction of short plugs of two different pseudostationary phases into the capillary. The separation was completed in less than 10 min. The method can be used in quantitative analysis. Linear correlation was obtained between concentration and peak area of 0.996 or better. The repeatability (RSD) of the compound peak areas ranged from 3.6% (methyltestosterone) to 6.2% (androstenedione). Limits of detection were between 73 microg/L (testosterone) and 160 microg/L (fluoxymesterone). As a demonstration of the method, androstenedione, testosterone and epitestosterone were determined in a spiked urine sample.     

Separation and online preconcentration by multistep stacking with large-volume injection of anabolic steroids by capillary electrokinetic chromatography using charged cyclodextrins and UV-absorption detection

The separation of three common anabolic steroids (methyltestosterone, methandrostenolone and testosterone) was performed for the first time by capillary EKC. Different charged CD derivatives and bile salts were tested as dispersed phases in order to achieve the separation. A mixture of 10 mmol/L succinylated-beta-CD with 1 mmol/L beta-CD in a 50 mmol/L borate buffer (pH 9) enabled the separation of the three anabolic steroids in less than 9 min. Concentration LODs, obtained for these compounds with low absorption of UV light, were approximately 5 x 10(-5) mol/L. The use of online reverse migrating sample stacking with large-volume injection (the effective length of the capillary) enabled to improve the detection sensitivity. Sensitivity enhancement factors (SEFs) ranging from 95 (for testosterone) to 149 (for methyltestosterone) were achieved by single stacking preconcentration. Then, the possibilities of multistep stacking to improve the sensitivity for these analytes were investigated. SEFs obtained by double stacking preconcentration ranged from 138 to 185, enabling concentration LODs of 2.79 x 10(-7) mol/L (for methyltestosterone), 3.47 x 10(-7) mol/L (for testosterone) and 3.56 x 10(-7) mol/L (for methandrostenolone). Although online triple stacking preconcentration was achieved, its repeatability was very poor and SEFs for the studied analytes were not calculated.     

Implementation of gas chromatography combined with simultaneously selected ion monitoring and full scan mass spectrometry in doping analysis

A comprehensive screening method for the detection of prohibited substances in doping control is described and validated. This method is capable of detecting over 150 components mentioned on the list of the World Anti-Doping Agency including anabolic androgenic steroids, stimulants and all narcotic agents that are currently analysed using different analytical methods. The analytes are extracted from urine by a combined extraction procedure using freshly distilled diethyl ether and tert-butyl methyl ether as extraction solvents at pH 9.5 and 14 respectively. Prior to GC-MS analysis the residues are combined and derivatised using a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, NH(4)I and ethanethiol. The mass spectrometer is simultaneously operated in the full scan mode (mass range varies along with GC-oven temperature program) and in the selected ion monitoring mode. The obtained limits of detection are in compliance with the requirements set by the World Anti-Doping Agency. Besides narcotics, stimulants and anabolic androgenic agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. This comprehensive screening method reduces the amount of urine needed and increases the sample throughput without a loss in sensitivity and selectivity.     

Large-Volume Sample Stacking with Polarity Switching in CE for Determination of Natural Polyphenols in Plant Extracts

A simple on-column preconcentration method for capillary electrophoretic determination of eight polyphenolic compounds (carnosic acid, cinnamic acid, caffeic acid and rosmarinic acid, quercetin, apigenin, luteolin and rutin) was devised. The method was applied for the assay of polyphenols in methanolic extract of the medicinal plant Orthosiphon Stamineus Benth. The analysis was carried out in fused silica capillaries (I.D. 50 μm, effective length 50 cm, total length 60 cm) with UV detection at 200 nm. The background electrolyte was 50 mM sodium tetraborate of pH 9.0 (adjusted with phosphoric acid). Large volume sample stacking with polarity switching was used for sensitivity enhancement. With sample injection representing 50% of capillary volume and polarity switching at 1.6 min, an average 90-fold enhancement of absorbance signal of the analytes was achieved. The calibration curves were linear (r = 0.9956–0.9994) in the range 0.2 to 1.8 μg mL^?1 of an analyte. The repeatability of migration times and peak areas was characterized by RSD values 0.11–0.57 and 1.63–5.66%, respectively. The proposed method offers favourable limits of detection (9–16 ng mL^?1) that compare well with those of LC.     

Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.     

Determination of Riboflavin in Cereal Grains by Capillary Electrophoresis with Laser-Induced Fluorescence Detection with On-Line Concentration

A very simple, sensitive, and reliable method for the routine determination of riboflavin in cereal grains by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was established. Two on-line concentration techniques, namely, stacking in reverse migrating micelles (SRMM) and sweeping, were examined to enhance the detection signal. The optimum separation buffer consisted of 20 mM phosphoric acid and 140 mM sodium dodecyl sulfate (SDS), and a hydrodynamic injection of 30 s at 0.5 psi was confirmed for the introduction of a sample. In addition, it was found that salt in sample matrix influenced the performance of SRMM, showing the standard addition method was required for the quantitative analysis. The linearity of the method was good with a range of 0.5–500 ng/mL (r = 0.9990). The limit of detection (LOD) was 0.29 ng/mL at a signal-to-noise ratio of 3. This procedure presented was successfully applied to determine riboflavin in 18 samples of 9 types of cereal grains. The recovery rates varied from 94% to 98%, and the relative standard deviation (RSDs) was less than 4.1%.     

Fast Screening for Tobacco-Specific N-nitrosamines by CZE Using Dynamically Coated Capillaries

In this paper, we propose a rapid capillary zone electrophoretic method with dynamically coated capillaries for separation of and screening for six tobacco-specific nitrosamines, a group of strong carcinogens found only in tobacco products. Dynamic coating of the capillary surface with a polycation coating (CElixir Reagent A) followed by a polyanion coating (CElixir Reagent B; pH 2.5) was accomplished by use of a commercially available reagent kit. With this method, six tobacco-specific nitrosamines: N??-nitrosonornicotine, N??-nitrosoanatabine, N??-nitrosoanabasine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitroamino)-4-(3-pyridyl)-1-butanol were simultaneously separated and determined within 3 min, with a satisfactory detection limit of 1.0 ??g mL^?1 for all the tobacco-specific nitrosamines at S/N = 3, and a linear relationship between UV absorbance and analyte concentration (R ² > 0.98). The repeatability of the method ranged from 0.24 to 0.73% (RSD, n = 5) for migration time and 0.25?C1.54% (RSD, n = 5) for peak area, demonstrating that this capillary electrophoretic method is better than conventional capillary zone electrophoresis.     

Trace analysis of acetylcholinesterase inhibitors with antipsychotic drugs for Alzheimer’s disease by capillary electrophoresis with on column field-amplified sample injection

A simple and sensitive capillary zone electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChEI), donepezil, and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid–liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in <20 min at 25 °C with a fused-silica capillary column of 60.2 cm?×?50 μm I.D. (effective length 50 cm) and a run buffer containing 120 mM phosphate (pH 4.0) with 0.1 % γ-cyclodextrin, 40 % methanol (MeOH), and 0.02 % polyvinyl alcohol as a dynamic coating to reduce analytes’ interaction with the capillary wall. Using phenformin as an internal standard (40.0 ng/mL), the linear ranges of the proposed method for the simultaneous determination of donepezil, rivastigmine, aripiprazole, quetiapine, risperidone, clozapine, ziprasidone, and trazodone were over the range 4.0–80.0 ng/mL, and olanzapine was over the range 1.0–20.0 ng/mL. The method was applied for concentrations monitoring of AChEIs and antipsychotic drugs in ten Alzheimer’s disease patients with behavioral and psychological symptoms of dementia after oral administration of the commercial products.

Detection of Nandrolone and Boldenone Abuse in the Ovine by GC�CMS�CMS

Under European legislation, the use of anabolic steroids as growth promoters in meat production is prohibited. Currently, there is no internationally accepted method used for the detection of the potentially endogenous steroids nandrolone and boldenone in the ovine. In the current study, a multi-residue GC?CMS?CMS-based urinary assay has been validated for boldenone as well as the nandrolone metabolites 5??-estrane-3??,17??-diol and epinandrolone. Using a standard addition calibration line approach in pooled bovine urine, the method was linear between the endogenous concentrations and those augmented with 6,000 pg mL^?1. The method was then applied to populations of wether (n = 242) and ewe (n = 237) ovine animals in order to establish urinary thresholds for detecting nandrolone and boldenone abuse. A statistical model (the Chebyshev inequality) was used to produce threshold concentrations for each analyte. Adjustment of the nandrolone metabolite data for specific gravity, a measure of the hydration status of the animal, allowed the effective thresholds to be reduced; potentially leading to a lower number of false positives. Furthermore, the proposed epinandrolone confirmatory thresholds (38,628 and 57,950 pg mL^?1 in wethers and ewes, respectively) were found to be effective in detecting abuse of nandrolone for at least 1 month post-dose of this steroid. However, further studies would be required to assess the efficacy of the proposed boldenone confirmatory thresholds (19,857 and 56,080 pg mL^?1 in wethers and ewes, respectively) since data on its excretion following administration to the ovine are lacking.     

High-sensitivity capillary electrophoresis method for monitoring purine nucleoside phosphorylase and adenosine deaminase reactions by a reversed electrode polarity switching mode

A simple, efficient, and highly sensitive in-line CE method was developed for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) present in membrane preparations of human 1539 melanoma cells. After filling the running buffer (50 mM borate buffer, 100 mM SDS, pH 9.10) into a fused-silica capillary (50 cm effective length × 75 μm), a large sample volume was loaded by hydrodynamic injection (5 psi, 36 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-20 kV). The current was monitored and the polarity was reversed when 95% of the current had been recovered. The separation of the neutral analytes (nucleosides and nucleobases) was performed by applying a voltage of 15 kV. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. For inosine and adenine detection limits as low as 60 nM were achieved. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analysis using CE with UV detection reported so far. The Michaelis-Menten constants (K(m)) for PNP and ADA and the inhibition constants (K(i)) for standard inhibitors determined with the new method were consistent with literature data.