取人尿液样品1.00 mL,加入1.0 mg·L^{-1 13}C-红霉素-d₃和阿莫西林-d₄的混合内标溶液10 μL和乙二胺四乙酸二钠(Na₂EDTA)-Mcllvaine缓冲液(pH 3.5±0.05) 0.30 mL,涡旋30 s,离心5 min,将上清液以流量1.0 mL·min^-1过活化好的Oasis HLB固相萃取柱,抽干柱子后用甲醇3 mL以1.0 mL·min^-1流量洗脱柱子,收集全部洗脱液,氮吹至近干,用10%(体积分数)甲醇溶液稀释至0.5 mL,涡旋混匀后过0.22 μm有机滤膜,滤液供超高效液相色谱-串联质谱仪分析。40种抗生素(8种磺胺类、6种大环内酯类、8种β-内酰胺类、4种四环素类、9种喹诺酮类、3种氯霉素类、1种林可酰胺类和1种喹喔啉类抗生素)在Kinetex^ F5 100A色谱柱(50 mm×3.0 mm,2.6 μm)上用不同体积比的0.1%甲酸(体积分数,下同)溶液和含0.1%甲酸的乙腈溶液的混合溶液进行梯度洗脱分离,用电喷雾离子源负(3种氯霉素类抗生素)、正离子模式电离,用多反应监测(MRM)模式检测,其中红霉素和阿莫西林采用内标法和基质匹配法定量,其他抗生素采用基质匹配法定量。结果表明,红霉素和青霉素V工作曲线的线性范围为1.0~100.0 μg·L^-1,其余38种抗生素工作曲线的线性范围为1.0~200.0 μg·L^-1,检出限(3S/N)为0.01~1.25 μg·L^-1;按标准加入法进行回收试验,回收率为51.0%~128%,测定值的相对标准偏差(n=5)为1.2%~14%。方法用于10份成年人尿液样品的分析,其中9份样品中检出了9种抗生素,检出量为0.09~322.86 μg·L^-1,其中阿莫西林检出率高达50%,检出量高达322.86 μg·L^-1。

An aliquot (1.00 mL) of human urine sample was taken, 10 μL of 1.0 mg·L^-1 mixed internal standard solution (containing ¹³C-erythromycin-d₃ and amoxicillin-d₄) and 0.30 mL of Na₂EDTA-Mcllvaine buffer solution (pH 3.5±0.05) were added. The mixed solution was vortexed for 30 s and centrifuged for 5 min. The supernatant was passed through Oasis HLB solid phase extraction column at flow rate of 1.0 mL·min^-1. The column was drained, and eluted by 3 mL of methanol at flow rate of 1.0 mL·min^-1. The eluate was collected, blown to near dryness by nitrogen, and diluted to 0.5 mL by 10% (volume fraction) methanol solution. The resulting solution was vortexed and passed through a 0.22 μm organic filter membrane, after which the filtrate was analyzed by ultra-high performance liquid chromatograph-tandem mass spectrometer. Forty antibiotics (8 sulfonamides, 6 macrolides, 8 β-lactams, 4 tetracyclines, 9 quinolones, 3 chloramphenicols, 1 lincosamide and 1 quinoxaline antibiotics) were separated by gradient elution with mixed solutions composed of 0.1% formic acid (volume fraction, the same below) solution and acetonitrile solution containing 0.1% formic acid at different volume ratios on Kinetex^® F5 100A column (50 mm×3.0 mm, 2.6 μm), ionized by negative (3 chloramphenicol antibiotics) and positive ion mode of electrospray ion source, and detected by MRM mode. Erythromycin and amoxicillin were quantified by internal standard and matrix matching method, and other antibiotics were quantified by matrix matching method. As shown by the results, linear ranges of working curves of erythromycin and penicillin V were 1.0-100.0 μg·L^-1, and linear ranges of working curves of the remaining 38 antibiotics were 1.0-200.0 μg·L^-1, with detection limits (3S/N) in the range of 0.01-1.25 μg·L^-1. Test for recovery was made by standard addition method, giving recoveries in the range of 51.0%-128%, and RSDs (n=5) of the determined values ranged from 1.2% to 14%. The proposed method was applied to the analysis of 10 adult urine samples, and 9 antibiotics were detected in 9 samples, with detection amounts in the range of 0.09-322.86 μg·L^-1, of which the detection rate of amoxicillin was as high as 50% and the detection amount of that reached up to 322.86 μg·L^-1.