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Abstract：
An analytical method combining high-throughput automatic solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine 16 antibiotics (macrolides, tetracyclines, quinolones, and sulfonamides) and 4 β-agonists (terbutaline, salbutamol, ractopamine, and clenbuterol) in human urine samples. After thawing at room temperature, 1 mL of urine was sampled and the internal standard was added, followed by the addition of 200 μL ammonium acetate buffer and 20 μL β-glucuronidase, and the mixture was incubated at 37 ℃ overnight. Automatic solid-phase extraction was used to extract the target compounds from the urine samples, and the recoveries were compared using different solid-phase extraction 96-well plates (PRiME MCX, Sep-Pak C18, PRiME HLB), types and volumes of rinse solutions and eluents. Satisfactory recoveries of the 20 target compounds were obtained using the Oasis PRiME HLB 96-well plate, with 1.5 mL 10% (v/v) methanol aqueous solution and 2.0 mL methanol as the rinse solution and eluent, respectively. The eluent was concentrated under nitrogen gas at 45 ℃, and the recoveries of the target compounds were compared under different conditions (completely or almost dry, drying to 1 mL, and adding water as a protective agent), and the recovery rate was optimal when water was added as a protective agent. In this study, two types of analytical columns (ACQUITY BEH C18 and ACQUITY HSS T3) and different gradient elution procedures and mobile phases were compared. The optimal chromatographic effect was realized using an HSS T3 column (100 mm×3.0 mm, 1.8 μm) and 0.1% (v/v) formic acid aqueous solution-0.1% (v/v) formic acid in acetonitrile as the mobile phase in gradient elution at a flow rate of 0.3 mL/min. Comparing the peaks observed using different proportions of methanol aqueous solution and the initial mobile phase as the injection solvent revealed that 30% (v/v) methanol aqueous solution was the optimal solution in terms of peak shape and signal-to-noise ratio. MS was conducted using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, and the MS parameters were optimized, including the curtain (CUR) and collision gases (CAD). The standard curve obtained using this method exhibited a good linearity (correlation coefficient>0.997), and the respective limits of detection and quantification were 0.02-0.12 ng/mL and 0.06-0.41 ng/mL. At spiked levels of 0.25, 2.5, and 12.5 ng/mL, the recoveries were in the range of 81.7%-120.0% (except that of tetracycline), the intra- and inter-day RSDs (n=6) were 1.1%-11.0% and 1.2%-13.0%, respectively. Azithromycin, trimethoprim, terbutaline, salbutamol, ractopamine, and clenbuterol displayed moderate matrix effects, but all targets exhibited weak matrix effects after correction using the isotope internal standard. To evaluate the accuracy of this method, BCR-503 (containing salbutamol and clenbuterol) and internal quality control samples were used and the concentrations of salbutamol and clenbuterol were within the reference ranges. Additionally, the mean concentrations of the 20 target compounds of two different internal quality control samples after 7 measurements were in the ranges of 0.44-0.59 ng/mL (0.5 ng/mL) and 1.72-2.16 ng/mL (2.0 ng/mL), respectively, which were satisfactory. In this study, the analytical method employed automatic sample pretreatment with a 96-well solid-phase extraction plate, and the detection efficiency was considerably improved. This method displays the advantages of simple operation, ideal recovery, a high sensitivity and weak matrix effect, which satisfies the requirements for the simultaneous determination of 16 antibiotics and 4 β-agonists in human urine samples. This study provides a crucial method for use in monitoring antibiotics and β-agonists in human urine and studying their exposure characteristics and health risks.
摘要：
开发了一种将高通量自动固相萃取与超高效液相色谱-串联质谱（UPLC-MS/MS）相结合的分析方法，以测定16种抗生素（大环内酯，四环素,喹诺酮类药物,和磺胺类药物）和4种β-激动剂（特布他林，沙丁胺醇,莱克多巴胺,和盐酸克伦特罗）在人体尿液样本中。在室温下解冻后，取尿样1mL,加入内标,然后加入200μL乙酸铵缓冲液和20μLβ-葡糖醛酸苷酶，并将混合物在37℃下孵育过夜。采用自动固相萃取法提取尿样中的目标化合物,并使用不同的固相萃取96孔板（PRiMEMCX，Sep-PakC18,PRiMEHLB),漂洗溶液和洗脱液的类型和体积。使用OasisPRiMEHLB96孔板获得了20种目标化合物的满意回收率，以1.5mL10%(v/v)甲醇水溶液和2.0mL甲醇作为冲洗液和洗脱液，分别。洗脱液在45℃氮气下浓缩，并比较了不同条件下目标化合物的回收率(完全或几乎干燥，干燥至1毫升，并加水作为保护剂)，当添加水作为保护剂时，回收率最佳。在这项研究中,比较了两种类型的分析柱(ACQUITYBEHC18和ACQUITYHSST3)以及不同的梯度洗脱程序和流动相。使用HSST3色谱柱（100mm×3.0mm，1.8μm）和0.1％（v/v）甲酸水溶液-乙腈中的0.1％（v/v）甲酸作为梯度洗脱的流动相，流速为0.3mL/min。比较使用不同比例的甲醇水溶液和初始流动相作为注射溶剂观察到的峰，发现就峰形和信噪比而言，30％（v/v）甲醇水溶液是最佳溶液。MS使用正电喷雾电离(ESI+)在多反应监测(MRM)模式下进行,并对MS参数进行了优化，包括窗帘（CUR）和碰撞气体（CAD）。使用该方法获得的标准曲线表现出良好的线性（相关系数>0.997），检测限和定量限分别为0.02-0.12ng/mL和0.06-0.41ng/mL。在0.25、2.5和12.5ng/mL的峰值水平下，回收率在81.7%-120.0%范围内(四环素除外)，日内和日间RSD(n=6)分别为1.1%-11.0%和1.2%-13.0%，分别。阿奇霉素,甲氧苄啶,特布他林,沙丁胺醇,莱克多巴胺,盐酸克伦特罗显示出中等的基质效应，但是在使用同位素内标校正后，所有目标都表现出微弱的基体效应。为了评估该方法的准确性，使用BCR-503(含有沙丁胺醇和克伦特罗)和内部质量控制样品,沙丁胺醇和克伦特罗的浓度在参考范围内。此外,7次测量后，两种不同内部质量控制样品的20种目标化合物的平均浓度在0.44-0.59ng/mL（0.5ng/mL）和1.72-2.16ng/mL（2.0ng/mL）的范围内，分别,这是令人满意的。在这项研究中,分析方法采用96孔固相萃取板自动样品预处理，检测效率大大提高。这种方法显示了操作简单的优点，理想的恢复，高灵敏度和弱基体效应，满足同时测定人尿样中16种抗生素和4种β-激动剂的要求。这项研究为监测人体尿液中的抗生素和β-激动剂以及研究其暴露特征和健康风险提供了至关重要的方法。
