Analysis of β-agonists and β-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry

A method using hollow fibre-protected liquid-phase microextraction (HF-LPME) with in situ derivatization followed by gas chromatography/mass spectrometry (GC/MS) was established for the analysis of β-agonists and β-blockers in urine. Because it can simultaneously extract and derivatize compounds of interest by methylbenzol and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) in HF-LPME, the approach overcomes the drawbacks of considerable time-consuming and tedious operation, meanwhile improves enrichment multiple. The optimized conditions were extraction for 20 min at 35 °C with 5.0 μL of mixed extraction solvent (methylbenzol/MSTFA = 1:1, v/v) with stirring speed of 925 rpm in 5.0 mL sample under pH 12.0 and 14% (w/v) NaCl. The method provided very wide linear ranges (0.25–400 ng mL⁻¹) and low detection limits in the range of 0.08–0.10 ng mL⁻¹ for clenbuterol, metoprolol and propranolol while enrichment factors reached up to 256. The analytes could be determined in spiked urine by the method with high extraction efficacy (93.79–109.04% recoveries) and precision (<9.70% RSD). It has a satisfactory result for metoprolol in practical human urine samples for a single-dose administration of 50 mg after 36 h. The proposed method only needs few microliters of organic solvent and derivatizing agent; the operation is simple, convenient and rapid for the trace analysis of β-agonists and β-blockers in biological fluids; it can be readily generalized for high sample throughput. So, it is hopeful that the study will facilitate the monitoring of β-agonists and β-blockers in the competition sports.     

固相萃取-气相色谱法测定动物组织中盐酸克伦特罗残留量

建立了固相萃取-气相色谱分析动物组织中盐酸克伦特罗残留量的方法．采用固相萃取,分离富集动物组织中的盐酸克伦特罗．肉样的加标回收率在70％～80％;最低检出限为1μg／kg．盐酸克伦特罗的硅烷化衍生产物,采用气相色谱(ECD检测器)检测,其衍生物的峰面积与样品浓度在0．005～1．0μg／mL范围内呈良好的线性关系,线性回归系数大于0．999．     

高效毛细管电泳对克伦特罗对映体的分离及定量检测

以羟丙基-β-环糊精(HP-β-CD)作为手性添加剂,采用毛细管区带电泳(CZE)成功分离了克伦特罗(Clenbuterol,Cle)对映体.研究了β-CD种类与浓度,缓冲液pH值及浓度,操作温度等对分离的影响.结果表明,以30 mmol/L的HP-β-CD为手性添加剂,50 mmol/L的磷酸盐缓冲溶液(pH 2.5)为缓中液,分离电压24 kV,操作温度20℃,可使Cle对映体实现基线分离,其分离度为6.78.对拆分机理也进行了探讨,测定了HP-β-CD与两对映体的结合常数及热力学参数;对CZE定量能力(线性,精度)进行了考察,R和S型的线性相关系数都大于0.998,两者的相对标准偏差(RSD)均低于2.2%.     

盐酸克伦特罗在纳米ZnO和多壁碳纳米管修饰玻碳电极上的电化学行为

制备了纳米ZnO与多壁碳纳米管(MWNTs)复合修饰玻碳电极(ZnO-MWNTs/GCE),考察了盐酸克伦特罗(CLB)在该修饰电极上的电化学行为。实验结果表明:纳米ZnO与MWNTs显著增强了修饰电极对盐酸克伦特罗的伏安响应,增加了电极的有效表面积,改善了电极的导电性和电催化活性。在2~30μmol·L-1和30~500μmol·L-1浓度范围内,CLB在所制备的修饰电极上的电流响应与其浓度线性关系良好,且该电极具有较好的重现性和稳定性。     

Solid substrate-room temperature phosphorimetry for the determination of residual clenbuterol hydrochloride based on the catalysis of sodium periodate oxidizing eosine Y

Clenbuterol hydrochloride (CLB) could catalyze NaIO₄ oxidation of eosine Y (R), which caused the room temperature phosphorescence (RTP) signal of R to quench sharply. The ΔI_P (=I_P2 − I_P1, I_P2 was RTP intensities of reagent blank and I_P1 was RTP intensities of test solution) of the system was directly proportional to the content of CLB. According to that academic thought, a new solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace CLB has been established. This method has high sensitivity (detection limit (LD): 0.021 zg spot⁻¹, corresponding concentration: 5.2 × 10⁻²⁰ g mL⁻¹) and good selectivity (Er = ±5%, interfering species were of no interference). It has been applied to the determination of residual CLB in the practical samples. The results were verified using HPLC and GC/MS methods. The reaction mechanism of catalytic SS-RTP for the determination of residual CLB was also discussed.     

A straightforward route to obtain C1-labeled clenbuterol

Singly-labeled ¹³C₁-clenbuterol has been synthesized employing a straightforward pathway starting from easily available acetanilide. The critical step was the Friedel-Crafts acylation of this compound with marked acetyl chloride, being the first example of the use of this methodology applied to the synthesis of a clenbuterol derivative. Subsequent chemical reactions afforded the desired product with good yields.     

Quantification of Clenbuterol in Human Plasma and Urine by Liquid Chromatography-Tandem Mass Spectrometry

The quantitative determination of clenbuterol in human plasma and urine is of particular interest for various fields such as clinical and forensic research and doping controls. A simple and rapid sample preparation procedure based on liquid-liquid extraction with subsequent re-extraction followed by liquid chromatography and electrospray ionization tandem mass spectrometry allowed the determination of clenbuterol in urine and plasma at detection and quantification limits of 0.1 ng mL⁻¹ and 0.2 ng mL⁻¹, respectively, with recoveries ranging from 85–96%. The fast and robust nature of the assay provides a rapid and cost-effective alternative to established procedures utilizing solid-phase extraction strategies.     

Three-phase solvent bar microextraction and determination of trace amounts of clenbuterol in human urine by liquid chromatography and electrospray tandem mass spectrometry

Three-phase solvent bar microextraction (TPSBME) technique is described for the quantitative determination of trace amounts of clenbuterol (CB) in urine samples using liquid chromatography (LC) and electrospray tandem mass spectrometry (ES-TMS). CB was extracted from a basified urine sample (donor phase) into the organic solvent residing in the pores of a freely moving hollow fiber and then back extracted into an acidic solution (acceptor phase) inside the lumen of the hollow fiber. The ends of the fiber were pressure-sealed. Here, forward and back extraction took place spontaneously. We studied various parameters affecting the extraction efficiency viz. type of organic solvent (octanol, nonanol and dihexyl ether) used for immobilization in the pores of the hollow fiber, i.e. extraction time (10-40 min), stirring speed (0-1000 rpm), effect of sodium chloride (0-25%, w/v) and concentration of the donor (0.25-3 M NaOH) and the acceptor (0.5-5 M formic acid) phases. After extraction, CB was analyzed by injecting the analyte enriched acceptor phase into LC combined with ES-TMS. Enrichment factor (79), repeatability (R.S.D. = 5.1%), correlation coefficient (0.9972, for the range of 0.1-4 ng mL⁻¹), detection limit (7 pg mL⁻¹) were also investigated. The present technique is compared with the reported solid phase microextraction techniques in terms of selectivity, analysis time per extraction, cost of analysis per extraction, and precision. Among all microextraction techniques reported, this technique is the most economical sample preparation/preconcentration technique to our knowledge. The method was applied for the analysis of CB in human urine.     

盐酸克仑特罗片剂中克仑特罗及其杂质含量的HPLC测定

建立了测定盐酸克仑特罗片剂含量的高效液相色谱法。选用Agilent Eclipse XDB-C8柱(4.6 mm×250 mm,5μm),流动相为甲醇-水(40∶60,含20 mmol.L-1磷酸二氢钾,以磷酸调至pH 4.0),流速为1.0 mL.min-1,检测波长240 nm,线性范围为0.50~50.0 mg.L-1(r=0.999 9),检出限为0.5 ng,回收率在98.5~102.0%之间,RSD为2.06%~3.07%(n=5)。     

Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of β-agonists in biological matrices

β-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 μg/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five β-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 μg/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.