Analysis of β-agonists by HPLC/ESI-MS(n) in horse doping control

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.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐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 β₂‐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 © 2009 John Wiley & Sons, Ltd.     

Determination of β-blockers and β-agonists using gas chromatography and gas chromatography-mass spectrometry--a comparative study of the derivatization step

There is a growing demand for the rapid screening of multiple β-blockers and β-agonists in a single analytical run in clinical toxicology, antidoping control, forensic and environmental science. Although GC-MS is very often used to determine pharmaceuticals from these groups of drugs, the literature data on the derivatization and MS analysis of mixtures of these compounds is limited. This paper compares and evaluates derivatization procedures for the determination of six β-blockers (acebutolol, atenolol, metoprolol, nadolol, propranolol, pindolol) and two β-agonists (salbutamol, terbutaline) using GC techniques. Nineteen different derivatizing reagents (nine of them used for the first time with almost all the drugs) were employed in order to obtain a single derivative for each target compound with the greatest effectiveness of this reaction. Trimethylsilylation, tert-butyldimethylsilylation, acylation (e.g. trifluoroacetylation), combined trimethylsilylation and acylation, and the formation of cyclized silyl derivatives were carried out and the mass spectra (EI, 70 eV) recorded. The influence of the reaction time and temperature on these procedures was investigated. Additionally, the effects of the type of solvent and the amount of added trimethylchlorosilane (TMCS) on the silylation of the target compounds using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were tested. Among of the five mentioned above derivatization procedures applied - trimethylsilylation was found to be the most effective for derivatizing the analytes. The best results were obtained with a 1:1 (v/v) mixture of 99% BSTFA+1% TMCS and ethyl acetate at 60 °C for 30 min. The MS data for different types of β-blocker and β-agonist derivatives is presented. The information in this paper is valuable for scientists working on the determination of β-blockers and β-agonists in biological and environmental matrices.     

气相色谱-质谱法测定动物组织中残留的10种β2-兴奋剂

采用同位素稀释法并结合固相萃取技术,建立了动物组织中马布特罗、特布他林、卡布特罗、克伦特罗、西马特罗、沙丁胺醇、clenpenterol、苯氧丙酚胺、班布特罗、莱克多巴胺等10种β2-兴奋剂残留的气相色谱-质谱(GC-MS)检测方法。动物组织中添加同位素内标D9-克伦特罗、D3-沙丁胺醇、D5-莱克多巴胺,经无水乙醇提取、正己烷脱脂、SLS离子交换固相萃取柱净化后,用双三甲基硅基三氟乙酰胺+1%三甲基氯硅烷衍生,再进行GC-MS测定,内标法定量。结果表明,动物组织中添加2.0～10.0 μg/kg水平的β2-兴奋剂,回收率为72.8%~110.3%,相对标准偏差为1.2%~11.3%,最低检测限为0.5～1.0 μg/kg。     

Electrochemical behavior of β2-agonists at graphite nanosheet modified electrodes

In this paper, the voltammetric characteristics of several β₂-agonists including salbutamol, ractopamine, bamethane, isoxsuprine, ritodrine, fenoterol, terbutaline, metaproterenol, clenbuterol, clenproperol, mabuterol, cimaterol, cimbuterol and brombuterol were comparatively evaluated using graphite nanosheet (GN) modified glassy carbon (GC) electrodes. All the compounds can be oxidized at GN modified electrodes with enhanced peak current and reduced peak potential compared with naked GC electrodes. The electrochemical behaviors of the compounds are different due to different substituent groups on the aromatic rings. For the first time, an ECE process was observed for salbutamol and its analogues. The capability of determining β₂-agonists individually or simultaneously from aqueous solution using differential pulse voltammetry and amperometry with the developed electrode was also investigated.     

基质固相分散/高效液相色谱-串联质谱法分析肉肠中4种β-2-受体激动剂残留

利用同位素稀释技术,建立了肉肠中4种β2-受体激动剂克伦特罗、莱克多巴胺、沙丁胺醇和特布他林的基质固相分散/高效液相色谱-串联质谱(MSPD/HPLC-MS/MS)分析方法。样品经C18填料研磨,甲醇洗脱,提取物经酶解后,用MCX小柱净化,经高效液相色谱分离,在正离子多反应监测(MRM)模式下用电喷雾电离串联质谱测定,内标法定量。4种β2-受体激动剂在0.2～20.0μg/L质量浓度范围内呈良好的线性关系,相关系数(r2)均大于0.990;沙丁胺醇和克伦特罗的检出限为0.10μg/kg;莱克多巴胺和特布他林的检出限为0.15μg/kg;方法的回收率为80%～109%,相对标准偏差小于10%。该方法简便快捷、灵敏度高,样品和溶剂用量少,可满足肉肠中4种β2-受体激动剂残留的快速检测。     

高效液相色谱-串联质谱法检测动物尿液中的15种β-受体激动剂

建立了高效液相色谱-串联质谱检测动物尿液中克仑特罗、莱克多巴胺、沙丁胺醇、西马特罗、马布特罗、妥布特罗、班布特罗、马贲特罗、塞布特罗、齐帕特罗、福莫特罗、氯丙那林、特布他林、喷布特罗和溴布特罗等15种β-受体激动剂的分析方法。样品用高氯酸溶液酸解及沉淀蛋白质,经HLB固相萃取小柱净化、富集后,以甲醇和0.1%(体积分数,下同)甲酸水溶液作为流动相进行梯度洗脱,采用多反应监测(MRM)模式进行定性和定量分析。15种β-受体激动剂在0.25～20 μg/L的范围内线性关系良好(r≥0.9995)。在0.25、1.0、10 μg/L添加水平下的回收率为62.1%～107%,相对标准偏差(n=10)为3.5%～9.9%,定量限(以信噪比＞10计)为0.25 μg/L。该方法精密度好,灵敏度高,能简便、快速、准确地测定动物尿液中的15种β-受体激动剂。     

Evaluation of matrix solid‐phase dispersion extraction for 11 β‐agonists in swine feed by liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of 11 β‐agonists (clenbuterol, salbutamol, ractopamine, terbutaline, fenoterol, cimaterol, isoxsuprine, mabuterol, mapenterol, clenproperol, and tulobuterol) in swine feed. This rapid, simple, and effective extraction method was based on matrix solid‐phase dispersion. The limit of quantification of clenbuterol, cimaterol, mabuterol, salbutamol, terbutaline, mapenterol, clenproperol, and tulobuterol was 1 μg/kg and that of ractopamine, fenoterol, and isoxsuprine was 2 μg/kg. The recoveries of β‐agonists spiked in swine feeds at a concentration range of 1–8 μg/kg were >83.1% with relative standard deviations <9.3%. This rapid and reliable method can be used to efficiently separate, characterize, and quantify the residues of 11 β‐agonists in swine feeds with advantages of simple pretreatment and environmental friendliness.     

UPLC–MS/MS测定动物源食品中9种β-受体激动剂

建立超高效液相色谱–串联质谱法检测动物源食品中克伦特罗、莱克多巴胺、沙丁胺醇、特布他林、氯丙那林、西马特罗、菲诺特罗、妥布特罗、喷布特罗等9种β-受体激动剂的方法。样品经β-葡萄糖醛苷酶酶解,用0.2mol/L乙酸铵溶液(pH 5.2)提取,阳离子固相萃取柱净化,以乙腈–0.2%甲酸水溶液作为流动相进行洗脱,用Eclipse Plus C_(18)(50 mm×2.1 mm,1.8μm)色谱柱分离,采用多反应监测(MRM)模式进行定性和定量分析。9种β-受体激动剂的质量浓度在0.1~10.0 ng/mL范围内与定量离子丰度呈良好的线性关系,线性相关系数均大于0.999,检出限为0.1μg/kg。平均回收率为85.0%~101.2%,测定结果的相对标准偏差为2.8%~9.5%(n=6)。该方法精密度好,灵敏度高,能简便、快速、准确地测定动物源食品中的9种β-受体激动剂。     

猪肝中β-受体激动剂多残留的样品前处理方法比较及同时检测

比较了乙酸铵提取法(即农业部1025号公告-18-2008方法)、乙腈直接提取法及10%碳酸钠-乙腈溶液提取法对猪肝脏中9种β-受体激动剂残留的提取效果。结果表明,乙酸铵提取法对非诺特罗和喷布特罗等药物的回收率较低(小于20%);乙腈直接提取法可有效提取出喷布特罗,其回收率达79%,但对非诺特罗的回收率仅为32%;而10%碳酸钠-乙腈溶液提取9种药物的效果明显好于其他两种方法,除了非诺特罗的回收率为72%外,其他8种药物的回收率均在80%以上。基于此,建立了猪肝脏中9种β-受体激动剂残留检测的高效液相色谱-串联质谱法。在优化条件下,9种药物在0.50～25μg/kg范围内线性关系良好,相关系数(r)均大于0.99;在0.50、2.0、10μg/kg 3个加标水平的回收率为71%～105%,相对标准偏差均小于15%;9种药物的检出限均达0.2μg/kg。方法的准确度和精密度达到残留分析要求。     

液相色谱-电喷雾串联质谱测定猪组织中β-兴奋剂残留的基质效应

系统评价了液相色谱-电喷雾串联质谱测定猪肌肉与肝脏组织中氯丙那林、西马特罗、特布他林、妥布特罗、沙丁胺醇、克伦特罗、喷布特罗、莱克多巴胺及非诺特罗9种β-兴奋剂残留的基质效应及其影响因素.以甲醇、乙腈及乙酸乙酯提取猪肝脏,9种分析物的基质效应分别为13.4％～47.0％、19.9％～63.2％及33.5％～69.6％.多因素方差分析表明,提取溶剂及猪组织来源是引起基质效应的主要因素;固相萃取小柱净化后,分析物浓度对肌肉与肝脏中β-兴奋剂(除莱克多巴胺)的基质效应影响显著;3种SPE小柱对猪组织中喷布特罗的基质效应影响差异显著.     

The analysis of beta-agonists by packed-column supercritical fluid chromatography with ultra-violet and atmospheric pressure chemical ionisation mass spectrometric detection

Packed-column supercritical fluid chromatography (pSFC) using ultra-violet (UV) and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS) provides a versatile method for the identification and quantification of beta-agonists. We have achieved good separation of clenbuterol, salbutamol, terbutaline and fenoterol with good resolution and reasonable retention times using a high concentration of methanol modifier in the supercritical CO2, together with small amounts of both acidic (trifluoroacetic acid, TFAA) and basic (triethylamine, TEA, or diethylamine, DEA) additives. APCI-MS gave unambiguous identification of the 4 analytes, and increasing cone voltage provided informative fragmentation patterns. The pSFC-MS technique was shown to be linear (R2 > or = 0.996) over the concentration range 1-50 micrograms ml-1. Single ion monitoring (SIM) gave detection limits (on-column) of 2.5 ng (clenbuterol), 0.83 ng (terbutaline), 7.6 ng (salbutamol) and 2.7 ng (fenoterol). The pSFC-MS system was shown to be reproducible within a day, between days, and between restrictors. Analysis of milk samples 'spiked' with beta-agonists showed that the matrix caused no interference, with detection limits of approximately 500 micrograms l-1 of beta-agonists. More dilute solutions could be analysed by pre-concentration before the SFC stage.     

Field-amplified on-line sample stacking for separation and determination of cimaterol, clenbuterol and salbutamol using capillary electrophoresis

A capillary electrophoresis method, using field-amplified sample injection (FASI), was developed for separation and determination of some beta 2-agonists, such as cimaterol, clenbuterol and salbutamol. The optimum conditions for this system had been investigated in detail. The precision of the migration time, peak height and accuracy were determined in both intra-day (n = 5) and inter-day (n = 15) assays. Under the optimum conditions, the detection limits (defined as S/N = 3) of this method were found to be lower than 2.0 ng/mL for all of these three beta 2-agonists, which were much lower than that of the conventional electro-migration injection method, the enhancement factors were greatly improved to be 30-40-fold. Such lower detection limit lets this method to be suitable for determination of above-mentioned beta 2-agonists in the urine sample. The mean recoveries in urine were higher than 96.2%, 95.6% and 95.3% for cimaterol, clenbuterol and salbutamol, respectively, with relative standard deviations lower than 3.5%.     

猪肉中β-受体激动剂AlphaLISA检测方法的建立

建立了快速检测猪肉中克伦特罗、沙丁胺醇、溴布特罗和特布他林4种β-受体激动剂的光激化学发光纳米均相时间分辨荧光免疫(Alpha LISA)分析方法。样品经乙酸铵和β-葡萄糖醛酸酶/硫酸酯酶酶解提取过柱,浓缩至干后,用缓冲溶液定容。样品与生物素化抗原、抗体、供体微珠和受体微珠进行酶联免疫,Alpha LISA进行检测,基质标准曲线定量。结果表明:沙丁胺醇、克伦特罗、溴布特罗和特布他林在0.1~500ng/m L范围内呈良好线性,相关系数均大于0.98,沙丁胺醇与克伦特罗、溴布特罗和特布他林的交叉反应率分别为143.3%,179.2%和95.6%,检出限为0.03~0.08 ng/m L。在0.5,10,20μg/kg 3个添加水平下,4种化合物的平均回收率为82.5%~115.9%,相对标准偏差(RSD)均小于12.0%。该方法操作简单,快速准确,适用于猪肉中β-受体激动剂的筛选与检测。     

Electrostacking online sample pre-concentration capillary electrophoresis with amperometric detection for beta2-agonists in human urine

A simple, rapid, sensitive and specific method using capillary electrophoresis (CE) coupled with electrostacking and amperometric detection (AD) has been developed for the simultaneous separation and determination of clenbuterol (CLB), terbutaline (TER), salbutamol (SAL) and formoterol (FMT). In this paper, the CE separation and AD conditions were investigated in detail. The optimum conditions were: pH 8.6 Na(2)B(4)O(7)-H(3)BO(3) buffer solution (20.0 mmol/L), 9 kV for the separation voltage, and 1000 mV (versus Ag/AgCl) for the detection potential. When the sample which was dissolved in 70% ACN-water mixture solution was injected 60 s by 15 kV electrokinetic injection, the best stacking effects was obtained. Under the optimum conditions, the enhancement factors of these beta(2)-agonists had been greatly improved more than 5500-fold compared with the conventional electrokinetic injection. And then an excellent linear response was obtained with LODs (S/N = 3) of 0.098, 0.024, 0.063 and 0.920 pmol/L for CLB, TER, SAL and FMT in urine, respectively. The precision was determined in both intra-day (n = 5) and inter-day (n = 15) assays, and the RSDs were not more than 2.1 and 3.4% for migration time and peak current, respectively. The proposed method has been applied to analyze human urine samples successfully.     

Analysis of exemestane and 17β-hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO-TMS derivatives

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17β-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50 ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10 ng/mL and 25 ng/mL, respectively.     

液相色谱-串联质谱结合谱库检索测定猪组织中的9种β-兴奋剂残留量

建立了猪组织中9种β-兴奋剂(克伦特罗、莱克多巴胺、沙丁胺醇、利托君、特布他林、班布特罗、妥布特罗、西马特罗和异舒普林)多残留的液相色谱-四极杆/线性离子阱串联质谱(QTrap LC-MS/MS)检测方法。样品经β-葡萄糖醛苷酶/芳基硫酸酯酶酶解提取,液液萃取净化,以乙腈-甲酸水溶液为流动相,经C18柱分离后用QTrap LC-MS/MS进行多反应监测(MRM)、信息相关采集(IDA)、增强子离子扫描(EPI)和谱库检索分析。9种β-兴奋剂的线性范围为0.1～50.0 μg/L,线性关系良好(r＞0.99);样品中9种目标物在0.5、1.0和5.0 μg/kg添加水平下的回收率为72.0%～95.1%,相对标准偏差为3.1%～12.1%;方法检出限为0.1～0.2 μg/kg。实际样品检测结果表明,本方法可实现猪组织样本中β-兴奋剂残留量的灵敏、准确的定性和定量分析。     

Cyclodextrins as chiral selectors in capillary electrophoresis: a comparative study for the enantiomeric separation of some beta-agonists.

A comparative study for the enantiomeric separation of terbutaline, clenbuterol, salbutamol and dobutamine was performed by capillary electrophoresis using cyclodextrins and their derivatives as chiral selectors. Several parameters such as buffer composition and temperature were studied. Simple, fast and reliable enantioseparations were achieved for all drugs studied, especially when the isomerically pure sulfated beta-cyclodextrin derivatives were used as chiral selectors.     

High-speed gas chromatography in doping control: fast-GC and fast-GC/MS determination of beta-adrenoceptor ligands and diuretics

In official doping controls, about 300 drugs and metabolites have to be screened for each sample. Moreover, the number of determinations to be routinely processed increases continuously as the number of both samples and potential illicit drugs keeps growing. As a consequence, increasingly specific, sensitive, and, above all, fast methods for doping controls are needed. The present study presents an efficient fast-GC/MS approach to the routine screening of two different classes of doping agents, namely beta-adrenoceptor ligands and diuretics (belonging to the S3, P2, and S5 groups of the WADA list of prohibited substances). Narrow bore columns (100 mm id) of different lengths and coated with apolar stationary phases were successfully used to separate the derivatized analytes; preliminary experiments (results not shown) showed better performances with OV-1701 for the separation of beta-adrenoceptor ligands. On the same stationary phase some diuretics required too high a temperature or a long isothermal time for elution, in which case a DB1-MS column was preferred. Two methods of sample preparation, derivatization, and analysis were used on aqueous standard mixtures of, respectively, (i) eight beta-adrenoceptor ligands, including five beta-antagonists (acebutolol, alprenolol, atenolol, metoprolol, pindolol) and three beta2-agonists (salbutamol, clenbuterol, terbutaline) and (ii) seventeen diuretic drugs (acetazolamide, althiazide, bendroflumethiazide, bumethanide, canrenone, chlorothiazide, chlortalidone, clopamide, ethacrinic acid, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, indomethacine, spironolactone, triamterene, trichloromethiazide) and one masking agent (probenecid). The mixture of beta-adrenoceptor ligand derivatives was efficiently separated in about 5.6 min, while the one of 18 diuretics and masking agents required less than 5 min for analysis. Limits of detection were from 1 microg/L for pindolol, ethacrinic acid, furosemide, indomethacine, and trichloromethiazide, to 20 microg/L for terbutaline, salbutamol, and metoprolol, and 50 microg/L for clopamide; the instrumental repeatability proved to be excellent (area RSD% <2 for almost all analytes). For this work a quadrupole MS with inert ion source has been used, demonstrating that the quadrupole technology is perfectly adequate to provide precise integration of 400 ms-wide GC peaks.     

超高效液相色谱-串联质谱法检测动物源性食品中残留的9种β-受体激动剂

建立了动物源性食品中特布他林、西马特罗、沙丁胺醇、非诺特罗、氯丙那林、莱克多巴胺、克仑特罗、妥布特罗和喷布特罗等9种β-受体激动剂残留检测的超高效液相色谱-串联质谱方法。样品经酶解后,用高氯酸去除蛋白质等杂质,调节上清液的pH值后,分别用乙酸乙酯和叔丁基甲醚进行萃取,再用MCX固相萃取柱净化,然后用Waters Acquity UPLC BEH C18色谱柱(50 mm×2.1 mm,1.7 μm)分离,以0.1%甲酸乙腈溶液和0.1%甲酸水溶液为流动相进行梯度洗脱,外标法定量。结果表明:9种β-受体激动剂在0.25～5 μg/kg的空白添加浓度范围内呈良好的线性关系,相关系数(r)均大于0.990;特布他林等8种药物的检出限为0.1 μg/kg,定量限为0.25 μg/kg;喷布特罗的检出限为0.25 μg/kg,定量限为0.5 μg/kg。从0.5,1和2 μg/kg共3个添加浓度的检测结果可以看出,9种药物的平均回收率为87.1%～108.6%,批内、批间相对标准偏差(RSD)均小于20%。该方法具有简便快捷、灵敏度高、定性准确等特点。