液相色谱串联质谱检测儿童患者血浆左氧氟沙星方法的建立及临床验证

Development of a LC-MS/MS method for the determination of levofloxacin in plasma from pediatric patients and validation of its clinical value

目的建立一种儿童患者左氧氟沙星血浆浓度的液相色谱串联质谱测定方法,并进行应用评估。方法前瞻性观察性研究。收集郑州大学附属儿童医院体检中心健康体检的儿童血浆标本作为空白血浆,以及5例呼吸科未使用左氧氟沙星、环丙沙星的儿童患者临床标本用于方法学验证。收集2023年10月1日至12月31日期间22例使用左氧氟沙星的儿童患者临床标本,男9例,女13例,年龄(8.1±3.7)岁,进行浓度测定。血浆样本经乙腈沉淀蛋白后,以环丙沙星为内标,采用液相色谱-质谱联用建立浓度测定方法。色谱柱为C18色谱柱(Shim-pac GIST-HP C18色谱柱,2.1 mm×100 mm,3μm);流动相A为0.1%甲酸水溶液,B为0.1%甲酸乙腈溶液,梯度洗脱,流速0.4 ml/min,柱温40℃,进样量1μl,总分析时间为9 min。质谱离子源为电喷雾离子源,设定为正离子模式,扫描方式为多反应监测模式,左氧氟沙星和环丙沙星的离子对分别为362.10→318.1和332.15→231.05。对方法进行特异性、灵敏度、线性、精密度、准确度、加样回收率、稳定性、基质效应、残留效应性能验证。使用SPSS 17.0统计分析数据,采用K-S检验检测变量的正态性。双侧检验,P<0.05为差异有统计学意义。结果血浆样品中左氧氟沙星在0.0625~20 mg/L线性关系良好(R^(2)>0.99),定量下限为0.0625 mg/L。在不同质控水平下,准确度为92.57%~104.39%,批内及批间精密度为2.32%~9.35%。正常血浆基质、5%溶血血浆基质、15%高脂血浆基质不影响左氧氟沙星血药浓度检测,短期存放样品的稳定性较好,残留不影响待测物的定量分析。共收集检测22例患者的34份临床标本,其中2份标本左氧氟沙星血药浓度测定结果低于定量下限,其余左氧氟沙星浓度为0.091~6.755 mg/L。C_(max)为(5.52±1.09)mg/L。结论本研究建立的血浆左氧氟沙星液相色谱串联质谱的方法分析性能符合要求,仅需血浆50μl,可用于儿童患者左氧氟沙星血药浓度检测。

Objective:To develop and validate a liquid chromatography-tandem mass spectrometry method for determining levofloxacin in plasma sample from pediatric patients.Method:This is a prospective,observational study.The clinical residual plasma samples from healthy individuals for physical examination in Children's Hospital Affiliated to Zhengzhou University were collected as blank matrix.Plasma samples from five pediatric patients who did not receive levofloxacin or ciprofloxacin in the department of Respiration were collected for methodological evaluation.In addition,34 clinical plasma samples from 22 pediatric patients(9 males and 13 females;mean age(8.1±3.7)years)using levofloxacin was collected,and their plasma concentrations were determined.Using ciprofloxacin as the internal standard,levofloxacin in plasma samples was quantified by liquid chromatography-tandem mass spectrometry following protein precipitation using acetonitrile.A C18 column(Shim-pac GIST-HP C18,2.1 mm×100 mm,3μm)and mobile phase composed of water(containing 0.1%formic acid)and acetonitrile(containing 0.1%formic acid)with gradient elution at a flow rate of 0.4 ml/min were used to separate levofloxacin.The column temperature was 40℃,injection volume was 1μl and the total analysis time was 9 min.Levofloxacin and ciprofloxacin were ionized with an ESI source in positive ion mode and detected in multiple reaction monitoring(MRM)mode.The detected ions of levofloxacin and ciprofloxacin were m/z 362.10→318.1 and 332.15→231.05,respectively.The method′s specificity,sensitivity,linearity,precision,accuracy,recovery rate,stability,matrix effect,and carry-over were validated.All statistical analyses were performed with SPSS statistical software(version 17.0).The normality of the data was detected by the K-S test.A P<0.05 was considered statistically significant for two tailed tests.Results:The LC-MS/MS method showed a good linearity within the range of 0.0625-20 mg/L,with the lower detection limit of levofloxacin of 0.0625 mg/L.The calibration curve for levofloxacin was Y=0.093X+0.010(R^(2)>0.99).Under different quality control levels,the accuracy ranged from 92.57%to 104.39%,and the intra-day and inter-day imprecision ranged from 2.32%to 9.35%.These values were not affected by the normal matrix,5%hemolysis matrix and 15%hyperlipidemia matrix.Furthermore,the levofloxacin plasma samples were stable in the short term.A total of 34 plasma samples from 22 patients were collected and analyzed.Only 2 plasma samples were below the lower limit of quantification,while the other plasma concentrations of levofloxacin were ranged from 0.091 to 6.755 mg/L.C_(max) was(5.52±1.09)mg/L.Conclusion:The LC-MS/MS method meets the requirements of the reference method and requires a small sample size(50μl),making it suitable for the determination of levofloxacin in plasma from pediatric patients.