Article

Analysis of β-agonists by HPLC/ESI-MSn in horse doping control

Authors:
  • LCH France
  • LCH, Verrières le Buisson, France
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Abstract

A sensitive method using LC/ESI-MS(n) has been developed on a quadrupole linear ion trap mass analyser for the detection of nine β(2) agonists (cimaterol, clenbuterol, fenoterol, formoterol, mabuterol, terbutaline, ractopamine, salbutamol and salmeterol) in horse urine. The method consists of solid-phase extraction on CSDAU cartridges before analysis by LC/ESI-MS(n) . The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allowed the detection of these compounds at pg/mL levels. Administration studies of fenoterol and formoterol are reported and show their possible detection after inhalation. The method is applicable for screening and confirmatory analysis.

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... Owing to very low limits of quantification, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the preferred technique for monitoring therapeutic levels of these drugs in biological matrices. Several methods for determination of fluticasone propionate and salmeterol have been published so far, with applications in pharmacokinetics (PK) or doping control (Laugher et al., 1999;Krishnaswami et al., 2000;Zhou et al., 2003;Van Eenoo et al., 2002;Lehner et al., 2004;Čapka and Carter, 2007;Carter and Čapka et al., 2008;Garcia et al., 2011). Most analytical methods presented in the literature deal separately with fluticasone propionate (Laugher et al., 1999;Krishnaswami et al., 2000) and salmeterol or groups of β-adrenergic receptor agonists (Van Eenoo et al., 2002;Lehner et al., 2004;Čapka and Carter, 2007;Garcia et al., 2011). ...
... Several methods for determination of fluticasone propionate and salmeterol have been published so far, with applications in pharmacokinetics (PK) or doping control (Laugher et al., 1999;Krishnaswami et al., 2000;Zhou et al., 2003;Van Eenoo et al., 2002;Lehner et al., 2004;Čapka and Carter, 2007;Carter and Čapka et al., 2008;Garcia et al., 2011). Most analytical methods presented in the literature deal separately with fluticasone propionate (Laugher et al., 1999;Krishnaswami et al., 2000) and salmeterol or groups of β-adrenergic receptor agonists (Van Eenoo et al., 2002;Lehner et al., 2004;Čapka and Carter, 2007;Garcia et al., 2011). For PK profiling of pharmaceutical formulations containing both active moieties, this separate approach is time-consuming; furthermore, previous methods often lack the sensitivity to detect pharmacologically relevant plasma concentrations, especially in the case of salmeterol. ...
Article
A new method for the fast simultaneous quantification of fluticasone propionate and salmeterol from plasma samples by liquid chromatography-tandem mass spectrometry, with adequate sensitivity for pharmacokinetic applications, was developed and validated. The chromatographic separation and mass-spectrometric parameters were optimized for the retention and detection of the two compounds, despite quite different structures and properties. Two columns connected in series were used, cation-exchange (Zorbax 300-SCX, 5 cm x 2.1 mm, 5 μm) and octadecyl (Discovery HSC₁₈, 10 cm x 2.1 mm, 5 μm). The mass-spectrometric interface was operated in negative electrospray ionization mode; high sensitivity and lesser matrix effects were obtained, permitting smaller consumption of plasma. The sample preparation was based on supported liquid-liquid extraction in 96-well format plates that provided clean samples with a simplified procedure that was suitable for automation. The method was validated according to regulatory guidelines, by assessing lower limits of quantification, selectivity, linearity, accuracy, precision, extraction recoveries and matrix effects. A comparison with two other methods for the separate determination of fluticasone propionate and salmeterol in plasma samples, previously developed by our group, is presented. The statistical evaluation of the results obtained with the three methods on a set of unknown samples from treated patients demonstrated good correlation (R² 0.987 for fluticasone propionate and 0.967 for salmeterol).
... β-agonists The ability of the strategy to identify the oral administration of clenbuterol was also tested in the validation scheme. Clenbuterol is approved for use in equine veterinary medicine to prevent bronchospasm, it also has repartitioning effects at higher doses and is not approved for such application (Garcia et al., 2011). The analysis of urine samples collected in Exp. ...
Article
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Introduction Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work focuses on assessing the robustness of the associated method and define its scope of application. Materials and methods Several hundred urine samples were selected from 14 different horses of ethically approved administration studies involving various doping agents’ (AAS, SARMS, β-agonists, SAID, NSAID) (328 urine samples). In addition, 553 urine samples from untreated horses of doping control population were included in the study. Samples were characterized with the previously described LC-HRMS/MS method, with the objective of assessing both its biological and analytical robustness. Results The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the misuse of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agents demonstrating complementary performances of traditional and omics approaches in the screening of anabolic agents in horses.
... The capacity of the strategy to highlight per os administration of clenbuterol was also included in the validation scheme. While clenbuterol is authorized in equine veterinary medicine to prevent bronchospasm, this substance also acts as a repartitioning agent at higher dose and is not authorized for such use (Garcia, 2011). The characterization of urine samples collected in the frame of Exp. ...
Preprint
Full-text available
Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work now focuses on assessing the robustness of the associated method and define its scope of application. Several hundred urine samples were selected from 16 different horses of ethically approved animal experiments involving various doping agents’ applications (AAS, SARMS, β -agonists, SAID, NSAID) (n = 349). In addition, 342 urine samples from untreated animals of general equine populations were included in the study. Samples were characterized with the previously described LC-HRMS/MS developed method, with the objective of assessing both its biological and analytical robustness’s. The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the administration of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agent’s residues, demonstrating complementarity performances of traditional and omics approaches in the screening of anabolic agents in horses.
... Various analytical methods based on LC-MS [14][15][16][17][18][19][20] , GC-MS [21,22] , internal extractive electrospray ionization-mass spectrometry (iEESI-MS) [23,24] , capillary electrophoresis with electrochemical detection [25] and immunoassay (EIA) [16,[26][27][28] are available for the detection of β-agonists. Since LC-MS/MS can rapidly and accurately quantify β-agonists in meat products [29] , we used LC-MS/MS to detect β-agonists in meat products. Clenbuterol, ractopamine, and salbutamol were selected as target analytes because they are the most commonly used β-agonists. ...
Article
Full-text available
This study was designed to investigate clenbuterol, salbutamol, and ractopamine concentrations in fresh meat products. A total of 801 samples of ribs, loin, and internal organs of pigs, cattle, and lamb were collected from retail and wholesale markets in nine districts of Jilin Province, Northeast China. The three β-agonists were analyzed by LC-MS/MS. Results showed that 11.36% of the samples contained prohibited β-agonists. Detection rates of β-agonists decreased in the following order: Clenbuterol (6.37%) > Ractopamine (2.62%) > Salbutamol (2.37%). The β-agonists levels were higher for pork than for beef and lamb. The detection rate of meat products decreased in the following order: pork loin (28.72%) > pork ribs (24.72%) > pork internal organs (20.24%) > beef loin (6.67%) > beef internal organs (6.19%) > beef ribs (5.00%) > lamb internal organs (3.45%) > lamb ribs (3.41%) > lamb loin (3.26%). Only one β-agonists was detected per sample. On the surface of our research, the amount β-agonists of added to fresh meat products in Jilin Province of China is relatively safe.
... At present, chromatographic analysis and immunoassays are the mainstream technologies for confirmation and screening of β-agonists in animal feed and animal products [7]. However, the various chromatographic methods reported, such as high-performance liquid chromatography (HPLC) [8,9], gas chromatography-mass spectrometry (GC-MS) [10,11], ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) [12,13] and capillary electrophoresis (CE) [14,15] are not suitable for field analysis because of the requirement of sophisticated instruments and complicated sample pretreatment. Therefore, plenty of immunoassays based on the enzyme-linked immunosorbent assay (ELISA) [16,17], electrochemistry [18], fluorescence [19], chemiluminescence [20] and surface-enhanced Raman scattering [21] have been prevalent methods recently and are particularly suitable for rapid screening. ...
Article
Interleukin-21 is a common γ-chain cytokine that controls the immune responses of B cells, T cells, and natural killer cells. Targeting IL-21 to strengthen the immune system is promising for the development of vaccines as well as anti-infection and anti-tumor therapies. However, the practical application of IL-21 is limited by the high production cost. In this study, we improved IL-21 production by codon optimization and selection of appropriate signal peptide in CHO-K1 cells. Codon-optimized or non-optimized human IL-21 was stably transfected into CHO-K1 cells. IL-21 expression was 10-fold higher for codon-optimized than non-optimized IL-21. We fused five different signal peptides to codon-optimized mature IL-21 and evaluated their effect on IL-21 production. The best result (a 3-fold increase) was obtained using a signal peptide derived from human azurocidin. Furthermore, codon-optimized IL-21 containing the azurocidin signal peptide promoted IFN-γ secretion and STAT3 phosphorylation in NK-92 cells similar to codon-optimized IL-21 containing original signal peptide. Collectively, these results indicate that codon optimization and azurocidin signal peptides provide an efficient approach for the high-level production of IL-21 as a biopharmaceutical.
... There are no data available on the detection of budesonide and betamethasone in faeces. Urinary levels detected in different animal species range from 0.02 ng/mL [29] to 10 ng/mL [30] for clenbuterol; from 0.05 ng/mL [12,27] . The extracted calibrators were injected just after being prepared, then kept at room temperature, 4°C, −20°C or at −80°C, and the same extract was again injected 7 days after preparation. ...
Article
The use of performance enhancing drugs is not only common in humans, but also in animal sports, including racing of horses, greyhounds and pigeons. The development of accurate analytical procedures to detect doping agents in sports is crucial in order to protect the fair-play of the game, avoid financial fraud in the attribution of eventual awards and, even more important, to protect the animals from harmful drugs and/or dangerous dosage regimens. The present study aimed to develop and validate, a method that enabled the screening and confirmation of the presence of a beta-agonist (clenbuterol) and three corticosteroids (betamethasone, prednisolone and budesonide) in faeces from pigeons. The extraction procedure entailed the combination of liquid-liquid extraction with solid-phase extraction and the analysis was performed by liquid- chromatography coupled to tandem mass spectrometry, with a single 15 minute chromatographic run-time. The method was validated concerning selectivity, linearity (with coefficients of determination always >0.99), accuracy (87.5-114.9%), inter-day and intra-day precisions, limits of detection (0.14-1.81 ng/g) and limits of quantification (0.49-6.08 ng/g), stability and extraction recovery (71.0%-99.3%). The method was successfully applied for the analysis of samples from two pigeons that had been orally administered betamethasone, demonstrating its suitability for doping control purposes.
... Thus, the development of a screening method that enables the sensitive and simultaneous identification of hundreds dissimilar analytes in these complex matrices is an uphill task. Several screening methods have been applied for the detection of groups of substances with similar properties and pharmacological activities (class based methods).[91][92][93][94][95][96][97][98][99]However, in recent years, research groups have shown a tendency to the establishment of comprehensive screening methodologies that provide broad coverage of a large number of different doping agents in equestrian sports. In general terms, the above purpose has been achieved by: ...
... Several screening methods have been applied for the detection of groups of substances with similar properties and pharmacological activities (class based methods). [91][92][93][94][95][96][97][98][99] However, in recent years, research groups have shown a tendency to the establishment of comprehensive screening methodologies that provide broad coverage of a large number of different doping agents in equestrian sports. In general terms, the above purpose has been achieved by: ...
... Several screening methods have been applied for the detection of groups of substances with similar properties and pharmacological activities (class based methods). [91][92][93][94][95][96][97][98][99] However, in recent years, research groups have shown a tendency to the establishment of comprehensive screening methodologies that provide broad coverage of a large number of different doping agents in equestrian sports. In general terms, the above purpose has been achieved by: ...
... Moreover, ␤ 2 -agonists were drugs that potentially produced a certain amount of anabolic-like effect so the World Anti-Doping Agency also restricted the use of ␤ 2 -agonists [6]. To comply with this legislation, various analytical methods have been developed to screen for ␤ 2 -agonist misuse [7][8][9][10], and these are now especially efficient for monitoring the illegal administration of ␤ 2 -agonists. However, because of a synergetic effect when combined with certain other molecules at lower doses, some ␤ 2 -agonist compounds may be used in combination with new unknown chemical structures (so-called "cocktails"), or with other ␤ 2 -agonist compounds, as illegal growth promoters in cattle or swine [11]. ...
Article
The illegal use of β2-agonists in livestock production was previously detected by efficient methods based on mass spectrometry to control the residues of these drugs. Nevertheless, such methods still remain a challenging task for authorities who monitor these residues because the use of "cocktails" composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention of illegal use of growth-promoting agents. Here, we outlined a metabolomics-based strategy for detecting the use of "cocktails" composed of mixtures of low amounts of three β2-agonists via urine profiling. Urine profiles of controls and swine treated with mixture of low amounts of three substances (clenbuterol, salbutamol, and ractopamine) were analyzed with ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The metabolic differences between controls and β2-agonists-treated groups were compared using multivariate data analysis. Fourteen metabolites were identified related with the β2-agonists treatment, while two co-biomarkers, 2-indolecarboxylic acid and fluorometholone acetate, either in single or "cocktails" of low-dose mixture of clenbuterol, salbutamol, and ractopamine, could be considered as diagnostic markers for the detection of illegal use of β2-agonists. The results of depletion study demonstrated that it is practical to use the markers for monitoring of β2-agonists. Copyright © 2015. Published by Elsevier B.V.
... The present study was based on five different methods of sample preparation, that is acetonitrile precipitation, membrane filtration, sample dilution with water by factors of five and 20, and proteinase K hydrolysis. The ability of proteinase K to digest native proteins [41] makes it useful for a variety of applications [42,43] and it is widely used in horse racing laboratories to digest the proteins occurring in urine [44]. Along with hydrolysis of peptide amides, dissociation of some noncovalent protein small-ligand bonds was an additional reason to consider and evaluate this method for metabolomic purposes. ...
Article
Background: Horse urine is the medium of choice for the implementation of metabolomic approaches aimed at improving horse doping control. However, drug analysis in this biofluid is a challenging task due to the presence of large amounts of interfering compounds. [br/] Methodology & Results: A comparative study of sample preparation has been conducted to evaluate five sample-preparation methods, namely acetonitrile precipitation, proteinase K hydrolysis, membrane filtration and sample dilution with water by factors of five and 20, for metabolome analysis using liquid chromatography coupled to high resolution mass spectrometry. Assessment was performed at both global and targeted levels, by using a few thousand features obtained from peak detection software, and internal standards and 100 annotated or identified metabolites. [br/] Conclusion: By considering the number of detected signals, their intensity and their detection repeatability, acetonitrile precipitation was selected as the most efficient sample-preparation method for the analysis of horse urine metabolome in liquid chromatography coupled to high resolution mass spectrometry conditions.
... Since the biotransformation pathway of β-agonists includes conjugation with glucuronic acid or sulfate, enzymatic hydrolysis using glucuronidase, arylsulfatase or both has been previously exploited to release their conjugates in urine [31,32,[41][42][43][44]. In this study, a solution of 30 μL containing 15 U/ml and 40 U/ml of glucuronidasi and arylsulfatase, respectively, yielded better results in the enzymatic deconjugation process. ...
Article
Full-text available
Ultra Performance Liquid Chromatography (UPLC) hyphenated to tandem Mass Spectrometry (MS/MS) was used for the development of an analytical method capable of simultaneous identification and quantification of 18 β-agonist compounds namely, brombuterol, chlorbrombuterol, cimaterol, cimbuterol, clenbuterol, clencyclohexerol, clenisopenterol, clenpenterol, clenproperol, hydroxymetylclenbuterol, isoxsuprine, mabuterol, mapenterol (clenbuterol-like compounds), ractopamine, ritodrine, salbutamol, salmeterol (salbutamol-like compounds) and zilpaterol in bovine urine. In compliance with the Commission Decision 2002/657/EC (CD 2002/657/EC), the method was validated applying a matrix-comprehensive in-house validation approach based on a fractional factorial design. Six experimental factors varied on two levels were selected for this purpose. The relevant validation parameters such as decision limit CCα (range, 0.24-0.51 μg L- 1) and detection capability CCβ (range, 0.27-0.71 μg L- 1), within-laboratory reproducibility (< 20%) as well as recovery (range, 92-109%) were in agreement with the performance criteria set in the CD 2002/657/EC. Overall, the proposed method enabled both screening and confirmatory detection of the β-agonist compounds in the framework of official monitoring plans.
... Conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a triple-quadrupole or ion-trap mass spectrometer has been applied successfully in doping control analysis in equine sports.1234567891011121314 Data acquisitions are usually done by selected reaction-monitoring or product-ion scanning mode allowing multiple targets to be covered in a single analysis. ...
Article
A recent trend in the use of high resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged due to significant improvement in high resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution, and mass stability. A number of HRAMS methods have been reported for the detection of multi‐drug residues in human or equine urine. As blood has become a common matrix for doping control analysis, especially in equine sports, a sensitive, fast and wide coverage screening method for detecting a large number of drugs in equine blood samples would be desirable. This paper presents the development of a liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) screening method for equine plasma samples to cover over 320 prohibited substances in a single analytical run. Plasma samples were diluted and processed by solid‐phase extraction. The extracts were then analyzed with LC‐HRMS in full‐scan positive electrospray ionization mode. A mass resolution of 60 000 was employed. Benzyldimethylphenylammonium was used as an internal lock mass. Drug targets were identified by retention time and accurate mass, with a mass tolerance window of ±3 ppm. Over 320 drug targets could be detected in a 13‐min run. Validation data including sensitivity, specificity, extraction recovery and precision are presented. As the method employs full‐scan mass spectrometry, an unlimited number of drug targets can theoretically be incorporated. Moreover, the HRAMS data acquired can be re‐processed retrospectively to search for drugs which have not been targeted at the time of analysis. Copyright © 2012 John Wiley & Sons, Ltd.
Article
Illegal use of ractopamine (RAC) in the food industry has dire consequences for health which should be curbed by inexpensive on-site checks. In this study, four advanced nanostructures of AuNPs were examined for this purpose. For the first time, a novel cost-effective colorimetric opto-sensor based on gold nanoparticles in aqueous solution was developed and successfully utilized for the recognition of RAC in real samples. The colorimetric chemosensor based on AuNPs-CysA exhibited a linear range of 0.1 μM to 0.01 M with a limit of detection (LOD) of 0.001 μM. Also, using AuNPs-DDT as a photonic probe two ranges of linearity of 0.01 to 50 μM and 0.005 to 0.01 M were obtained (LOD = 1 nM). The outstanding features of the utilized nanostructures are the simple preparation, the suitable stability of AuNPs-CysA and the excellent selectivity of AuNPs-DDT toward RAC recognition. Finally, the engineered colorimetric systems were combined with a simple and inexpensive optimized microfluidic glass fiber-based device. This work paves the way for devising inexpensive and efficient on-site recognition devices for food safety checks.
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In the present study, an antibody against phenylethanolamine A (PEA) was produced, confirmed, and used in a surface plasmon resonance (SPR)-based measurement. Bovine serum albumin (BSA)-conjugated PEA was linked to nano-gold particles bound to L-cysteine modified on the surface of a Nu-NP sensor chip. The concentrations of antigen and antibody were optimized, and the designed biosensor chip was investigated to examine the stability and accuracy of the proposed method. The recovery of PEA ranged from 80.4%-93.4% in swine urine samples with spike levels of 5, 10 and 20 ng/mL, and the relative standard deviations of PEA were less than 2%. PEA analogues, such as clenbuterol, ractopamine, and salbutamol, did not influence the PEA measurement. The developed method could be used to measure PEA in swine urine samples
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Despite the impressive innate physical abilities of horses, camels, greyhounds or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti‐doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports, over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices, published from 1990 to 2019 were considered. A total of 317 studies were included, of which 298 were related to horses, demonstrating significant advances towards the development of doping detection methods for equine sports. However, analytical methods for the detection of doping agents in sports involving other species are lacking. Due to enhanced accuracy and specificity, chromatographic analysis coupled to mass spectrometry detection is preferred over immunoassays. Regarding biological matrices, blood and urine remain the first choice, although alternative biological matrices, such as hair and faeces, have been considered. With the increasing number and type of drugs used as doping agents, the analytes addressed in the published papers are diverse. It is very important to continue to detect and quantify these drugs, recognizing those that are most frequently used, in order to punish the abusers, protect animals’ health, and ensure a healthier and genuine competition.
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Salmeterol is a long acting beta2‐agonist (LABA) widely used for treatment of airways disease. There is evidence that beta2‐agonists, including salmeterol, have the potential for performance enhancing effects when delivered at supratherapeutic doses. For this reason, all beta2‐agonists are currently on the Prohibited List issued by the World Anti‐Doping Agency (WADA) regardless of dosing route with some exemptions for inhaled salbutamol, formoterol, and salmeterol when used at therapeutic inhaled doses. For 2020, salmeterol use is permitted up to a therapeutic dosing threshold of 200 μg daily, but unlike salbutamol and formoterol, there is an anomaly; currently there is no urine threshold to control for supratherapeutic dosing beyond this dosing threshold. Salmeterol, however, is reportable as an Adverse Analytical Finding (AAF) at levels above 10 ng/ml. Complicating matters is that following inhalation, salmeterol parent drug is present at relatively low levels compared to other beta2‐agonists due to rapid metabolism to the metabolite, alpha‐hydroxysalmeterol, which is typically present at higher levels than parent drug. Moreover, peak parent drug levels following permitted therapeutic dosing are below the minimum required performance level (MRPL) of 10 ng/ml for salmeterol (50% of the MRPL that analytical laboratories are required to meet for non‐threshold beta2‐agonists), hence the presence of salmeterol may be unreported. For consistency, a urine threshold should be introduced for salmeterol as matter of priority, to balance the needs of athletes that use salmeterol therapeutically up to the agreed dosing threshold, with the need to control supratherapeutic dosing for doping intentions and athlete harm minimisation.
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In this work, an ion selective electrode modifying with graphene oxide (GO) was developed to determinate BAM by electrochemical method. First of all, composites of electro-active complex and GO was prepared as GO@BAM-TPB through drop-by-drop operation under stirring circumstance. In the next step, a membrane solution for electrode was made by mixing GO@BAM-TPB and film-forming materials and the prepared membrane solution was dipped uniformly onto the surface of bare platinum electrode. A few days later, the modified electrode had been dried spontaneously at room temperature, that could be reserved for use. Adequate characterizations have been done including not only structure and morphology of materials but also electrochemical properties of the modified electrode. We discussed modifying outcomes in both structure and morphology aspects. The parameters of modified electrode were as follows: a lower LOD (3.4×10-7 mol/L), a wider response range (1.0×10-1 to 1.0×10-6 mol/L), and a smaller response time (15s) than the traditional electrodes. Moreover, the modified electrode exhibits good selectivity, reproducibility, lifetime (up to 12 weeks) and pH range (3.5-7.1). Finally, BAM selective electrode was employed for determining BAM in real samples (eg, swine urine), as results shows, it proved to be a reasonable method to the determination of BAM.
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A fast analytical method for the simultaneous detection of 24 β2-agonists in human urine was developed and validated. The method covers the therapeutic drugs most commonly administered, but also potentially abused β2-agonists. The procedure is based on enzymatic deconjugation with β-glucuronidase followed by SPE clean up using mixed-phase cartridges with both ion-exchange and lipophilic properties. Instrumental analysis conducted by UHPLC-MS/MS allowed high peak resolution and rapid chromatographic separation, with reduced time and costs. The method was fully validated according ISO 17025:2005 principles. The following parameters were determined for each analyte: specificity, selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, matrix effect, recovery and carry-over. The method was tested on real samples obtained from patients subjected to clinical treatment under chronic or acute therapy with either formoterol, indacaterol, salbutamol, or salmeterol. The drugs were administered using pressurized metered dose inhalers. All β2-agonists administered to the patients were detected in the real samples. The method proved adequate to accurately measure the concentration of these analytes in the real samples. The observed analytical data are discussed with reference to the administered dose and the duration of the therapy.
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This chapter provides some updated information on contemporary methods for hormone and &;#x003B2;&;#x02010;agonist analyses. It deals with the classical approaches for the effective detection and identification of exogenous hormones. The chapter examines specific problems related to control strategies for natural hormones. These include both the traditional and generally recognized natural hormones as well as a series of androgenic steroids that can be present in biological samples obtained from a series of species. The fact that natural background concentrations can be present strongly complicates the analyses. The application of mass spectrometry (MS) in combination with gas chromatography (GC) or liquid chromatography (LC) is considered the &;#x00022;gold standard&;#x00022; for analytical methods in residue analysis. The chapter presents an overview of the available bio&;#x02010;based screening methods for the detection of hormones and &;#x000DF;&;#x02010;agonists, focusing on estrogens, androgens, progestogens, corticosteroids, thyroids, &;#x000DF;2&;#x02010;agonists, and growth hormones (GH).
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Due to the lack of sensitivity in current methods for the determination of fenoterol (Fen), a rapid LC-MS/MS method was developed for the determination of (R,R')-Fen and (R,R';S,S')-Fen in plasma and urine. The method was fully validated and was linear from 50pg/ml to 2000pg/ml for plasma and from 2.500ng/ml to 160ng/ml for urine with a lower limit of quantitation of 52.8pg/ml in plasma. The coefficient of variation was <15% for the high QC standards and <10% for the low QC standards in plasma and was <15% for the high and low QC standards in urine. The relative concentrations of (R,R')-Fen and (S,S')-Fen were determined using a chirobiotic T chiral stationary phase. The method was used to determine the concentration of (R,R')-Fen in plasma and urine samples obtained in an oral cross-over study of (R,R')-Fen and (R,R';S,S')-Fen formulations. The results demonstrated a potential pre-systemic enantioselective interaction in which the (S,S')-Fen reduces the sulfation of the active (R,R')-Fen. The data suggest that a non-racemic mixture of the Fen enantiomers may provide better bioavailability of the active (R,R')-Fen for use in the treatment of cardiovascular disease.
Article
A liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) method was established for the simultaneous determination of the levels of 10 β2-agonists in meat. The samples were extracted using an aqueous acidic solution and cleaned up using a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) technique utilising a DVB-NVP-SO3Na sorbent synthesised in-house. First, the β2-agonist residues were extracted in an aqueous acidic solution, followed by matrix solid-phase dispersion for clean-up. The linearities of the method were R(2)=0.9925-0.9998, with RSDs of 2.7-15.3% and 73.7-103.5% recoveries. Very low limits of detection (LOD) and quantitation (LOQ) of 0.2-0.9μg/kg and 0.8-3.2μg/kg, respectively, were achieved for spiked meat. The values obtained were lower than the maximum residue limits (MRLs) established by the EU and China. These results clearly demonstrate the feasibility of the proposed approach. The evaluated method provided reliable screening, quantification and identification of 10 β2-agonists in meat. Copyright © 2015 Elsevier Ltd. All rights reserved.
Article
In this study a one-step immunochromatographic assay based on competitive format was developed for the rapid detection of phenylethanolamine A (PEAA) residues in urine and pork samples. A monoclonal antibody against PEAA was produced from BALB/c mice immunized with the PEAA-BSA conjugate. The results of this qualitative test strip were to be interpreted visually. The visual detection limit (VDL) and threshold level of the optimized immunochromatographic assay for PEAA were 0.1 ng/mL and 0.5 ng/mL, respectively. Cross-reactions with other β-agonists were not significant inhibitions to the performance of the test strip assay. The results from the test strip were in a good agreement with those obtained using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay. The immunochromatographic assay developed here was a useful on-site screening tool that is rapid to use, low in cost, and extremely convenient for the detection of PEAA in urine samples and pork samples. © 2015 Institute of Food Technologists®
Article
A salbutamol-bovine serum albumin and a salbutamol-ovalbumin coating antigen were synthesized, and six New Zealand white rabbits were treated with the immunogen to obtain polyclonal antibodies to develop a rapid, sensitive, and indirect chemiluminescent enzyme immunoassay (ciCLEIA) for the analysis of swine and bovine urine. The prepared antibodies showed high cross-reactivities with clenbuterol (139.6%) and brombuterol (225%). Under the optimized conditions, the linear dynamic range and the limit of detection (LOD) for salbutamol were from 0.007 to 0.17 µg L−1 and 0.003 µg L−1, with a correlation coefficient of 0.9965 and a half maximum inhibition concentration of 0.028 µg L−1. Recoveries for salbutamol, clenbuterol, and brombuterol were from 78.8% to 119.0% with intra-assay and inter-assay coefficients of variation less than 13.9% and 19.7%, respectively. The reported ciCLEIA was about 10-fold more sensitive for salbutamol and 20-fold more sensitive for clenbuterol compared to conventional methods. This study showed that ciCLEIA was a reliable, convenient, and sensitive method for the simultaneous determination of salbutamol, clenbuterol, and brombuterol in swine and bovine urine.
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As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC-MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC-MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC-MS to support protein quantification in drug discovery.
Article
This paper describes a reserved-phase HPLC method for detecting clenbuterol (CB) and ractopamine (RP) using an isocratic solvent-free mobile phase. The separations were performed a Kaseisorb® LC C1-300-5 (100 × 4.6 mm, 5 μm) with 0.05 mol/L octane sulfonic acid mobile phase and a photodiode array detector. The total run time was <5 min. The system suitability was well within the international acceptance criteria. The detection limits were 0.68 ng/mL for CB and 1.24 ng/mL for RP, respectively. A harmless method for simultaneous detecting CB and RP was developed and may be further applied to the quantification in foods.
Article
In the present study a general screening protocol was developed to detect prohibited substances and metabolites for doping control purposes in equine sports. It was based on the establishment of a unified sample preparation and on the combined implementation of liquid and gas chromatographic MS analysis. The sample pretreatment began with two parallel procedures: enzymatic hydrolysis of sulfate and glucuronide conjugates, and methanolysis of the 17β-sulfate steroid conjugates. The extracts were treated for LC-TOF-MS, GC-HRMS and GC-MS assays. The majority of the prohibited substances were identified through a high mass accuracy technique, such as LC-TOF-MS, without prior derivatization. The sample preparation procedure included the formation of methylated and trimethylsilylated derivatives common in toxicological GC-MS libraries. The screening method was enhanced by post-run library searching using automated mass spectral deconvolution and identification system (AMDIS) combined with deconvolution reporting software (DRS). The current methodology is able to detect the presence of more than 350 target analytes in horse urine and may easily incorporate a lot of new substances without changes in chromatography. The full scan acquisition allows retrospective identification of prohibited substances in stored urine samples after reprocessing of the acquired data. Validation was performed for sixty representative compounds and included limit of detection, matrix interference - specificity, extraction recovery, precision, mass accuracy, matrix effect and carry over contamination. The suitability of the method was demonstrated with previously declared positive horse urine samples.
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Formoterol is a new threshold substance in the prohibited list 2012 according to World Anti-Doping Agency. Extracted by ethyl acetate using formoterol-D6 as internal standard, formoterol underwent a constant flow rate gradient elution separation in reversed-phase liquid chromatography. Subsequently, mass spectrometry analysis was conducted by tandem mass spectrometry in the multiple reaction monitoring mode. According to the proposed method, a calibration curve was constructed in the range of 0.2–500 ng/mL (r2 = 1) with a limit of quantification 0.2 ng/mL. The mean extracted recovery assessed at three different concentrations (1, 30 and 100 ng/mL) was more than 80%. The method was validated by the analysis of three quality control samples from World Association of Anti-Doping Scientists. In conclusion, the developed and validated method was sensitive, accurate and precise for the quantification of formoterol in human urine for doping control purposes.
Article
The separation of β-agonists was performed for the first time on a silica-based weak cation exchange (WCX) stationary phase in which aqueous electrolyte solution with small amount of organic solvent was used as the mobile phase. Compared to the organic solvent used in reverse phase HPLC mode, the merits of using an aqueous mobile phase include unique selectivity, environment-friendly operation and reduced operation costs. The effective separation of four commonly used β-agonists including salbutamol, terbutaline, metoprolol and ractopamine, were achieved with the mobile phase of 20 mM ammonium formate/acetonitrile (5%). The interference from the hydrophobic analytes could be easily eliminated due to their poor retention under this mode. The detection limits for the above analytes when fluorescence detection is employed were in the range of 3.55–8.32 μg kg−1 and the spiked recoveries for metoprolol for the spike sample reached about 83% and the relative standard deviation (RSD) was 4.7%.
Article
A new method has been developed using a hybrid triple-quadrupole linear ion trap (QTrap) mass spectrometer for the fast detection and identification of nine beta-agonists, clenbuterol, salbutamol, ractopamine, ritodrine, terbutaline, isoxsuprine, tulobuterol, cimaterol and bambuterol, in one single liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The homogenized tissue samples were purified with liquid-liquid extraction after enzymatic hydrolysis by P-glucuronidase/aryl sulfatase. After gradient elution separation on C(18) LC column using acetonitrile and formic acid aqueous solution as the mobile phases, a multiple reaction monitoring (MRM) scan as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information dependent acquisition (IDA) experiment. Finally, the identification of the drugs was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The analytical method in the present study was well validated and good results were obtained with respect to precision, repeatability and spiked recovery. The limits of detection of residues were 0.1 -0.2 micro g/kg for beta-agonists, and with a linear range from 0.1 to 50.0 micro g/L. Three concentration levels of 0. 5, 1. 0 and 5. 0 pg/kg were spiked in pig tissues, and the overall recoveries were between 72.0% and 95.1% with the relative standard deviations (RSDs) between 3. 1% and 12.1%. The real sample test showed that this method could be used for sensitive and accurate determination of beta-agonist residues in pig tissue
Article
An online stacking capillary electrophoresis (CE) method, cation-selective exhaustive injection sweeping MEKC (CSEI-sweep-MEKC), is developed and optimized for analysis of ractopamine (RP) and its homolog dehydroxyractopamine (DRP) in porcine meat. Chemometric experimental design was used to achieve the best possible optimization and reduce the number of trials and errors. The CSEI-sweep-MEKC method enables ng/g-level analysis, with limits of detection (LODs) in meat of 5 ng/g for RP and 3 ng/g for DRP (S/N = 3). A higher conductivity buffer (HCB) zone was injected into the capillary, allowing the analytes to be electrokinetically injected at a voltage of 9 kV for 12 min. Using 125 mM of SDS and 15% of MeOH in the sweeping buffer, RP and DRP were well separated. The method was validated with a linear calibration curve of 10-300 ng/g (r > 0.994). In comparison with the normal CZE method (1 psi, 10 s), this stacking strategy resulted in 900 times sensitivity enhancement. This technique was further applied for analyzing 7 kinds of commercial meats and the residual ractopamine was detected in one (5.76 ng/g of RP). The data was corresponding to that analyzed by the commercial testing kit and MS-spectra. This method was successfully used on real samples and is considered feasible for serving as a tool of routine examination in markets.
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A heart-cut two-dimensional high-performance liquid chromatography method for enantiomeric determination of salbutamol, salmeterol and atenolol in urine is presented. It involves the use of two separations in a liquid chromatography–liquid chromatography achiral–chiral coupling. Target compounds were previously separated in a primary column (KinetexTM HILIC, 2.6 lm, 150 9 2.1 mm I.D.) with a mixture of MeOH:ACN:ammonium acetate buffer (5 mM, pH 6) 90:5:5 (v/v/v) as mobile phase at a flow rate of 0.40 mL min-1. Enantiomeric separation was carried out by transferring peak of each compound through a switching valve to a vancomycin chiral column (Chirobiotic TM V, 2.6 lm, 150 9 2.1 mm I.D.) using MeOH:ammonium acetate buffer (2 mM, pH 4) 97:3 (v/v) as mobile phase at a flow rate of 0.50 mL min-1. Ultraviolet detection was done at 227 nm. The method was applied to determine target analytes in urine samples after enzymatic hydrolysis with b-glucuronidase from Helix pomatia, followed by a solid-phase extraction procedure using Isolute� HCX mixed-mode cartridges. Extraction recoveries ranged from 82 to 90 % in urine samples. Detection limits were 0.091–0.095 lg for each enantiomer of atenolol and between 0.058 and 0.076 and 0.18–0.14 lg for enantiomers of salbutamol and salmeterol, respectively (3 mL of urine). Linearity ranges were between 0.5 and 10 lg mL-1. Intraday and interday reproducibilities of enantiomeric ratio and enantiomeric fraction, expressed as relative standard deviation, were between 1.9 and 9.0 %. The optimized method was successfully applied to the analysis of urine samples obtained from excretion studies in volunteers and in freezedried urine samples, containing urinary components with MW\10,000 and components with MW[10,000, spiked with different amounts of studied drugs.
Article
A simple and efficient method based on a novel solid phase extraction (SPE) cartridge and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) was developed for the determination of clenbuterol residue in pork. The minced pork sample was ultrasonically extracted by 5% (v/v) perchloric acid and centrifuged at 10 000 r/min for 15 min, then the supernatant was purified by an SMCX cartridge, which is a novel SPE column based on homemade silica matrix with two mixed modes of reversed-phase and strong cation exchange, for the selective enrichment and purification of the analyte. The linear range of the method was 0.25 - 50 microg/kg with a correlation coefficient of 0.998 2. The average recoveries ranged from 62.2% to 72.0% at the three spiked levels of 1.25, 12.5 and 50 microg/kg with the relative standard deviations (RSDs) between 4.2% and 6.1%. The limit of detection (S/N = 3) was 0.05 microg/kg. The method is simple, fast and can be extended to enrich and determine beta-agonist drugs.
Article
The synergistic influences of analyte concentration, sample source, and solid-phase extraction (SPE) type on matrix effects in the multiresidue analyses of eight β-agonists with LC-ESI-MS/MS were evaluated. Porcine muscle and liver extracts and urine from diverse sources were purified by strong or mixed-mode cation exchange and molecularly imprinted polymer SPE cartridges, respectively. Three spiked concentrations (2, 10, and 20 ng/mL) of eight β-agonists in the purified matrices and the different sample sources were analyzed. The results show that for most β-agonists there are significant differences in matrix effects between analyte concentrations or sample sources (P < 0.05), whereas there is no significant difference in matrix effects between different SPE cartridges (P > 0.05). Results from main effects testing indicated that analyte concentration was the main effector.
Article
A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C(18) reversed-phase column, eluted with mobile phase of acetonitrile-ammonium acetate (5 m m; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers.
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Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection and confirmation of 38 singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine after solid-phase extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase extraction (SPE) using an ISOLUTE(®) CBA SPE column and analysed by LC/MS/MS in the positive electrospray ionisation mode. Separation of the 38 QADs was achieved on a polar group embedded C18 LC column with a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and confirmation of the 38 QADs at sub-ppb to low-ppb levels in equine urine could be achieved within 16 min using selected reaction monitoring (SRM). Matrix interference of the target transitions at the expected retention times was not observed. Other method validation data, including precision and recovery, were acceptable. The method was successfully applied to the analyses of drug-administration samples.
Article
A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS) method was developed and validated for the determination of Tramadol in human plasma and urine. The analyte was separated on a Diamonsil C18 column with ammonium acetate (5 mmol x L(-1))-methanol (50:50,v:v) adjusted PH by caustic soda at a flow rate of 0.8 ml min(-1), and analyzed by mass spectrometry is in positive ion mode. The ion mass spectrum of m/z were 264.1 for Tramadol and 248.0 for Tinidazole (I.S.), respectively. The weighted (1/x2) calibration curve was linear over plasma concentration range 1.00-400.00 ng/ml and urine concentration range 0.01-16.00 microg/ml, with a correlation coefficient (r) of 0.9995 and 0.9997, respectively. The lower limit of quantification in human plasma was 1.00 ng/ml. The inter-and intra-day precisions (CV%) in both plasma and urine were lower than 10%, the mean method accuracies and recoveries from spiked plasma samples at three concentrations ranged from 98.2 to 100.1% and 61.6 to 62.9%, respectively. The developed method was successfully applied to determine Tramadol in human plasma and urine, and provided suitable profiles for clinical pharmacokinetic study of Tramadol.
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Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches.
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We have investigated the detection, confirmation, and metabolism of the beta-adrenergic agonist ractopamine administered as Paylean to the horse. A Testing Components Corporation enzyme-linked imunosorbent assay (ELISA) kit for ractopamine displayed linear response between 1.0 and 100 ng/mL with an I-50 of 10 ng/mL and an effective screening limit of detection of 50 ng/mL. The kit was readily able to detect ractopamine equivalents in unhydrolyzed urine up to 24 h following a 300-mg oral dose. Gas chromatography-mass spectrometry (GC-MS) confirmation comprised glucuronidase treatment, solid-phase extraction, and trimethylsilyl derivatization, with selected-ion monitoring of ractopamine-tris(trimethylsilane) (TMS) m/z 267, 250, 179, and 502 ions. Quantitation was elaborated in comparison to a 445 Mw isoxsuprine-bis(TMS) internal standard monitored simultaneously. The instrumental limit of detection, defined as that number of ng on column for which signal-to-noise ratios for one or more diagnostic ions fell below a value of three, was 0.1 ng, corresponding to roughly 5 ng/mL in matrix. Based on the quantitation ions for ractopamine standards extracted from urine, standard curves showed a linear response for ractopamine concentrations between 10 and 100 ng/mL with a correlation coefficient r > 0.99, whereas standards in the concentration range of 10-1000 ng/mL were fit to a second-order regression curve with r > 0.99. The lower limit of detection for ractopamine in urine, defined as the lowest concentration at which the identity of ractopamine could be confirmed by comparison of diagnostic MS ion ratios, ranged between 25 and 50 ng/mL. Urine concentration of parent ractopamine 24 h post-dose was measured at 360 ng/mL by GC-MS after oral administration of 300 mg. Urinary metabolites were identified by electrospray ionization (+) tandem quadrupole mass spectrometry and were shown to include glucuronide, methyl, and mixed methyl-glucuronide conjugates. We also considered the possibility that an unusual conjugate added 113 amu to give an observed m/z 415 [M+H] species or two times 113 amu to give an m/z 528 [M+H] species with a daughter ion mass spectrum related to the previous one. Sulfate and mixed methyl-sulfate conjugates were revealed following glucuronidase treatment, suggesting that sulfation occurs in combination with glucuronidation. We noted a paired chromatographic peak phenomenon of apparent ractopamine metabolites appearing as doublets of equivalent intensity with nearly identical mass spectra on GC-MS and concluded that this phenomenon is consistent with Paylean being a mixture of RR, RS, SR, and SS diastereomers of ractopamine. The results suggest that ELISA-based screening followed by glucuronide hydrolysis, parent drug recovery, and TMS derivatization provide an effective pathway for detection and GC-MS confirmation of ractopamine in equine urine.
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In order to investigate the detection of boldenone in horse mane samples, a boldenone study was conducted on two horses. The analytical procedure consisted in a hydrolysis using the Sorensen buffer, a liquid-liquid extraction using diethyl ether and a PFPA derivatization. The instrumental method was a gas chromatography sequential mass spectrometry performed on an ion trap instrument in full scan mode. The limit of detection was estimated to 1 pg mg−1. The detection of boldenone in the mane was made possible for up to 12 months after administration.
Article
A fast screening protocol was developed and validated for the simultaneous determination of 15 beta(2)-agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid-liquid extraction, followed by derivatization of the extract and detection of beta(2)-agonists trimethylsilyl-derivatives by fast-gas chromatography/electron impact-mass spectrometry (fast-GC/EI-MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra- and inter-assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast-GC/MS sequences, based on the use of short columns, high carrier-gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright (c) 2009 John Wiley & Sons, Ltd.
Article
A method for the screening and confirmatory analysis of beta-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen DAU or Bond Elut Certify cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography-mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14-120 h depending on the drug.
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Gas chromatographic and mass spectrometric properties of cyclic methyl and n-butyl boronates of 13 beta blockers and bronchodilators were investigated for potential use in screening and quantitation in biological materials of interest. The tested compounds included acebutolol, atenolol, labetalol, levobunolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, albuterol, and isoproterenol. The cyclic methyl boronates were superior in gas chromatographic properties to the corresponding n-butylboronates of these compounds. Higher weights of fragments obtained with n-butyl boronation may be advantageous for analyses of comRlex biological matrices. Mass spectra and retention times relative to SKF-525A are provided.
Article
Recent reports on the misuse of beta 2-agonists, both as stimulants and as "anabolic agents" in sports, highlight the importance of screening and confirmation methods for these compounds in anti-doping control procedures. Although only a few analytical methods have been developed for this purpose, the large experience gained, both in pharmacokinetic studies and above all in the control of the residues of beta 2-agonists in animal fluids and tissues, can be of great help in the anti-doping field. This paper reviews single-residue (SR) and multi-residue (MR) methods developed for the analysis of beta 2-agonists in urine, plasma and hair samples, based on hyphenated chromatographic and mass spectrometric techniques, published in the last ten-year period. The evolution from GC-MS analysis after derivatization, with particular attention to the features of different proposed derivatives, to the most recent applications of coupled-column liquid chromatography (LC-LC) combined with tandem mass spectrometric detection (MS-MS) via a thermospray (TSP) interface is illustrated, and future perspectives in the field are outlined.
Article
Clenbuterol is a beta2 agonist/antagonist bronchodilator marketed as Ventipulmin and is the only member of this group of drugs approved by the US Food and Drug Administration (FDA) for use in horses. Clenbuterol is a class 3 drug in the Association of Racing Commissioners International (ARCI) classification system; therefore, its identification in postrace samples may lead to sanctions. Recently, the sensitivity of postrace testing for clenbuterol has been substantially increased. The objective of this study was to determine the 'detection times' for clenbuterol after administration of an oral clinical dose (0.8 g/kg, b.i.d.) of Ventipulmin syrup. Five horses received oral clenbuterol (0.8 g/kg, b.i.d.) for 10 days, and urine concentrations of clenbuterol were determined by an enhanced enzyme-linked immunoabsorbent assay (ELISA) test and gas chromatography/mass spectrometric (GC/MS) analysis by two different methods for 30 days after administration. Twenty-four hours after the last administration, urine concentrations of apparent clenbuterol, as measured by ELISA, averaged about 500 ng/mL, dropping to about 1 ng/mL by day 5 posttreatment. However, there was a later transient increase in the mean concentrations of apparent clenbuterol in urine, peaking at 7 ng/mL on day 10 postadministration. The urine samples were also analysed using mass spectral quantification of both the trimethylsilyl (TMS) and methane boronic acid (MBA) derivatives of clenbuterol. Analysis using the TMS method showed that, at 24 h after the last administration, the mean concentration of recovered clenbuterol was about 22 ng/mL. Thereafter, clenbuterol concentrations fell below the limit of detection of the TMS-method by day 5 after administration but became transiently detectable again at day 10, with a mean concentration of about 1 ng/mL. Derivatization with MBA offers significant advantages over TMS for the mass spectral detection of clenbuterol, primarily because MBA derivatization yields a high molecular weight base peak of 243 m/z, which is ideal for quantitative purposes. Therefore, mass spectral analyses of selected urine samples, including the transient peak on day 10, were repeated using MBA derivatization, and comparable results were obtained. The results show that clenbuterol was undetectable in horse urine by day 5 after administration. However, an unexpected secondary peak of clenbuterol was observed at day 10 after administration that averaged approximately 1 ng/mL. Because of this secondary peak, the detection time for clenbuterol (0.8 g/kg, b.i.d. x 10 days) is at least 11 days if the threshold for detection is set at 1 ng/mL.
Article
An experimental approach for the validation of chromatographic qualitative methods and its application in an antidoping control laboratory is described. The proposed strategy for validation of qualitative methods consists of the verification of selectivity/specificity, limit of detection (LOD), extraction recovery and repeatability (intra-assay precision). A one-day assay protocol, based on the analysis of five blank samples obtained from different sources and four replicates of control samples at two different concentrations of the analytes, has been defined to evaluate the validation parameters. The following evaluation criteria have been applied: absence of interfering substances at the retention time of the analytes in the blank samples to check the selectivity/specificity of the method, the LOD recommended by international sports authorities has to be attained, and for repeatability, the relative standard deviation should be <25% for the low concentration control sample and <15% for the high concentration control sample. Qualitative screening procedures are able to detect a great number of analytes so that extraction and analysis conditions are always a compromise for the different analytes. For this reason, no minimum acceptance criteria have been defined for data of extraction recoveries. The proposed protocol has been used for the validation of the screening and confirmation qualitative methods included in the scope of the accreditation of an antidoping control laboratory according to ISO quality standards.
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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.
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A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the limit of detection was 0.125 ng/mL. The excretion profile was determined after administration of 500 microg salmeterol (Serevent) to four standard-bred mares via a metered dose inhaler (MDI) with an Equinehaler adapter. Salmeterol was detected from 1 h until 12 h post-administration. Maximum urinary concentrations varied between 2.3 and 14.9 ng/mL.
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Clenbuterol, a beta-adrenergic agonist, is used in the treatment of recurrent airway obstruction in horses. It is prohibited by horse racing authorities, because of its stimulating and growth-promoting properties. However, information on detection times of clenbuterol after administration by nebulization is lacking. In this study, a fast, sensitive quantitative GC-MS(2) method for the detection of clenbuterol in urine was developed. Alkaline liquid-liquid extraction was followed by derivatization to a cyclic methyl boronate derivative and analysis on a Finnigan MAT GCQ instrument. Method validation showed good linearity in the range 0.1-2.0 ng/mL, excellent repeatability and specificity. The limit of quantitative detection of the method was 0.1 ng/ml. Different instrumental parameters of the ion trap mass spectrometer were changed to increase the number of diagnostic ions for the cyclic methyl boronate derivative of clenbuterol. The influence of these changes and their applicability within the requirements and the criteria for mass spectrometry set by the responsible regulatory bodies are discussed. Clenbuterol was administered via nebulization to five standardbred mares (0.4 micro g/kg body weight). Analysis of the urine samples resulted in the detection of clenbuterol, as early as 2 h post administration and for up to 36 h post treatment. Generally, maximum urinary concentrations of 1.2 ng/mL were reached after -6-9 h.
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Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine specimens, acidic re-extraction of the analytes. In addition, a degradation product of formoterol caused by acidic hydrolysis during sample preparation could be identified and utilized as target compound in screening and also confirmation methods. The screening procedures cover 18 or 19beta(2)-agonists, the estimated limits of detection of which for equine and human urine samples vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively. A single LC/MS/MS analysis can be performed in 9 min.
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A GC-MS procedure for the detection of different beta-agonists in urine samples based on two consecutive derivatization steps is described. The derivatization procedure is based on the consecutive formation of cyclic methylboronate derivatives followed by a second derivatization step with MSTFA on the same extract, forming TMS derivatives. Injections in the GC-MS system may be carried out after each one of the derivatization steps, obtaining enough information for unambiguous identification. Limits of detection for the two derivatization steps ranged from 0.5 to 5 ng/ml. This procedure was tested with the beta-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol, alpha-hydroxy-salmeterol and terbutaline.
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Ion suppression, a matrix effect that affects quantitative mass spectrometry, is one of the main problems encountered in liquid chromatography/tandem mass spectrometry. Two different clean-up steps for the multi-residue analysis of beta-agonists in urine were evaluated with respect to minimisation of ion suppression, namely, a mixed-phase solid phase extraction (SPE) column, i.e., clean screen Dau (CSD), and a molecular imprinted polymer (MIP) SPE column. Ion suppression experiments revealed that CSD sample clean-up can lead to false negative results for some beta-agonists, and that clean-up using MIP columns is more selective for beta-agonists than the use of CSD columns.
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The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.
View this article online at wileyonlinelibrary.com Copyright ©
  • P Garcia
P. Garcia et al. Biomed. Chromatogr. 2011; 25: 147–154 View this article online at wileyonlinelibrary.com Copyright © 2010 John Wiley & Sons, Ltd.