| 引用本文: | 宋小霞,白玉廷,秀仁高娃,毛伟,斯琴塔娜,李培锋,曹金山.液质联用-同位素内标法测定赛马血液、尿液中茶碱残留[J].中国现代应用药学,2021,38(11):1332-1338. |
| SONG Xiaoxia,BAI Yuting,XIUREN Gaowa,MAO Wei,SIQIN Tana,LI Peifeng,CAO Jinshan.Determination of Theophylline Residues in Racehorse Serum and Urine by Liquid-mass-isotope Internal Standard Method[J].Chin J Mod Appl Pharm(中国现代应用药学),2021,38(11):1332-1338. |
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| 液质联用-同位素内标法测定赛马血液、尿液中茶碱残留 |
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宋小霞1,2, 白玉廷1,2, 秀仁高娃1,2, 毛伟1,2,3, 斯琴塔娜1,4, 李培锋2,3, 曹金山2,3
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1.内蒙古农业大学马违禁药品检测实验室, 内蒙古 010018;2.内蒙古农业大学兽医学院, 内蒙古 010018;3.内蒙古农业大学农业部动物疾病临床诊疗技术重点实验室, 内蒙古 010018;4.内蒙古农业大学理学院, 内蒙古 010018
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| 摘要: |
| 目的 建立液质联用-同位素内标法测定赛马血液和尿液中茶碱残留的方法。方法 采用6D-茶碱作为内标物，利用强阳离子固相萃取小柱提取茶碱。采用Poroshell 120 SB-C18色谱柱（2.1 mm×50 mm，2.7 μm），0.1%甲酸水溶液（5 mmol·L–1甲酸铵）-0.1%甲酸乙腈为流动相，梯度洗脱；流速为0.4 mL·min–1；电喷雾源（ESI）离子源；正离子模式；茶碱以181.1>124.1，96.0 m/z（内标物187.1>126.9，99.0 m/z）以多反应监测（MRM）扫描方式进行定性定量。结果 使用Waters Oasis PRIME MCX 3CC/60 mg固相萃取小柱最优，血清和尿液茶碱浓度在1.00~100.00 ng·mL–1，2.50~100.00 ng·mL–1与峰面积线性关系良好（r2分别为0.999 9和0.999 6），检测限为0.30，0.75 ng·mL–1，定量限为1.00，2.50 ng·mL–1，相对回收率分别为98.93%~114.49%、94.85%~116.25%，批内精密度≤3.81%，批间精密度≤15.53%，血清基质对茶碱有较强抑制作用，尿液抑制作用较弱。结论 经方法学验证，该方法操作简单、快速、准确、灵敏度高，可适用于马兴奋剂中茶碱残留量的检测。 |
| 关键词: 茶碱 液质联用 兴奋剂 血液 尿液 |
| DOI：10.13748/j.cnki.issn1007-7693.2021.11.009 |
| 分类号:R917.101 |
| 基金项目:内蒙古自治区重大专项（201602053）；内蒙古农业大学引进优秀博士人才科研启动项目（RZ1900003570） |
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| Determination of Theophylline Residues in Racehorse Serum and Urine by Liquid-mass-isotope Internal Standard Method |
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SONG Xiaoxia1,2, BAI Yuting1,2, XIUREN Gaowa1,2, MAO Wei1,2,3, SIQIN Tana1,4, LI Peifeng2,3, CAO Jinshan2,3
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1.Inner Mongolia Agricultural University, Equine Anti-doping Laboratory, Hohhot 010018, China;2.Inner Mongolia Agricultural University, College of Veterinary Medicine, Hohhot 010018, China;3.Inner Mongolia Agricultural University, Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry Agriculture and Rural Affairs, P. R. China, Hohhot 010018, China;4.Inner Mongolia Agricultural University, College of Science, Hohhot 010018, China
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| Abstract: |
| OBJECTIVE To establish a method for the determination of theophylline residues in racehorse serum and urine by liquid-mass-isotope internal standard method. METHODS The strong cation exchange solid phase extraction was used to extract theophylline combined 6D-theophylline as internal standard. The Poroshell 120 SB-C18(2.1 mm×50 mm, 2.7 μm) was chosen, with 0.1% formic acid(containing 5 mmol·L–1 ammonium formate)-0.1% formic acid acetonitrile by gradient elution. The flow rate was 0.4 mL·min–1. Electrospray source(ESI) ion source was used. Multi-reaction positive mode was used and ion pair of 181.1>124.1, 96.0 m/z and 187.1>126.9, 99.0 m/z were used for qualitative of theophylline and internal standard respectively. RESULTS Waters Oasis PRIME MCX 3CC/60 mg solid phase extraction column was optimal. The concentrations of serum and urine ranged 1.00–100.00 ng·mL–1 and 2.50–100.00 ng·mL–1 had a good linear relationship with the peak area(r2 were 0.999 9 and 0.999 6, respectively). The detection limits were 0.30, 0.75 ng·mL–1, and the quantitative limits were 1.00, 2.50 ng·mL–1. The relative recoveries were 98.93%–114.49% and 94.85%–116.25%, respectively. The intra-batch precision RSD was≤3.81%, and the inter-batch precision RSD was ≤15.53%. The serum matrix had a strong inhibitory effect on theophylline, and the inhibition effect of urine was weak. CONCLUSION The method was proved to be simple, rapid, accurate and sensitive, and can be applied to the determination of theophylline residue in horse race doping control samples. |
| Key words: theophylline LC-MS/MS doping serum urine |
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