Simultaneous determination of Methotrexate,Levetiracetam,and Lamot-rigine in small volume of plasma by HPLC-MS/MS and its clinical applica-tion
Objective To established a HPLC-MS/MS method for determination the concentration of Methotrexate,Levetiracetam,and Lamotrigine in small volume of plasma and to monitor therapeutic drugs in children.Methods Drug-containing plasma 10 μl was obtained,and the samples were pretreated by protein precipitation.The chromatography was performed on Agilent C18 column(50 mm×2.1 mm,1.8 μm),the mobile phase was 0.1%formic acid and methanol,and gradient elution;the flow rate was 0.4 ml/min.Electrospray ion source positive ion mode was used,ions monitored were m/z 455.1→308.2 for Methotrexate,m/z 458.1→311.1 for Methotrexate-D3(internal standard),m/z 171.1.4→126.2 for Levetiracetam,m/z 177.2→132.2 for Levetiracetam-D6(internal standard),and m/z 256.1→211 for Lamotrigine,m/z 262.1→217.1 for Lamotrigine-13C3-D3(internal standard),respectively.Results Methotrexate had a good linear relationship between 25-1 500 ng/ml,the standard curve equation was Y=0.251 6X+0.003 4(r=0.998 9),the lower limit of quantitation was 25 ng/ml;Levetiacetam had a good linear relationship between 1-50 μg/ml,the standard curve equation was Y=16.687 6Y+0.000 2(r=0.991 1),and the lower limit of quantitation was 1 μg/ml;Lamotrigine has a good linear relationship between 0.5-25.0 μg/ml,the standard curve equation was Y=46.369 5X-0.059 9(r=0.999 1),the lower limit of quantitation was 0.5 μg/ml.The accuracy within and between batches and precision of samples met the requirements,and the matrix effect met the requirements.This method was fully verified and successfully applied to therapeutic drug monitoring of 1 348 pediatric patients.Conclusion The method is simple,convenient,and sensitive,and can be applied to the analysis of Methotrexate,Levetiracetam,and Lamotrigine in human plasma,and is suitable for the monitoring of therapeutic drugs in children.
MethotrexateLevetiracetamLamotrigineHPLC-MS/MSTherapeutic drug monitoring
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