自主研制四极杆-线形离子阱质谱仪测定血清中甲氨蝶呤

Determination of Methotrexate in Serum by House-Made Quadrupole-Linear Ion Trap Mass Spectrometer

本研究基于自主研制的QLIT-6610MD液相色谱-四极杆-线形离子阱-质谱联用仪,建立了准确测量血清中甲氨蝶呤的同位素稀释质谱法。采用甲醇沉淀血清样品的蛋白后测定上清液。通过基质匹配标准曲线定量,在10~5000μg/L浓度范围内,甲氨蝶呤的线性系数R^(2)>0.9990,定量限为10μg/L,质控品精密度优于6.94%,血清样本添加回收率为86.39%~97.84%,变异系数(CV)≤8.04%。应用QLIT-6610MD与AB QTRAP 6500+液相色谱-质谱联用仪同时测定70例临床血清样本,并采用Pearson系数与BLand-ALtman法进行比较。结果表明,2台仪器所测结果一致,QLIT-6610MD可达到三重四极杆质谱仪定量测定血清中甲氨蝶呤的水平,能够满足临床诊断需求,为临床质谱分析提供了一种新的国产仪器选择。此外,本研究还初步探讨了多级碎裂技术(MSn)测定血清中甲氨蝶呤的可能性,为后续研究奠定基础。

Methotrexate(MTX)is a therapeutic drug that is widely used in clinic for treatment of a variety of cancers.However,MTX has limited selectivity and serious cytotoxicity,at the same time,there are problems such as narrow therapeutic window and large individual variability in metabolism and excretion of MTX.Therefore,blood concentration monitoring is required for MTX administration in clinical practice.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)has the advantages of strong specificity,good separation characteristics,high sensitivity and fast detection speed,which has become the internationally recognized“gold standard”for blood concentration monitoring of drugs.In this study,an isotope dilution mass spectrometry method was developed for accurate measurement of MTX in serum by the house-made QLIT-6610MD quadrupole-linear ion trap liquid chromatography-mass spectrometer.Unlike the traditional triple quadrupole QqQ tandem mass spectrometer,the quadrupole-linear ion trap(Q-LIT)mass spectrometer adopts the quadrupole-linear ion trap direct axial coupling design,i.e.,a single linear ion trap is used to replace the QqQ tandem mass spectrometer’s back-end fragmentation cell and the quadrupole,and no other components are installed between the quadrupole and linear ion trap,which greatly streamlines the quality and efficiency of the mass spectrometer while ensuring the original performance of the instrument.With this unique configuration,the QLIT-6610MD instrument uses a simultaneous fragmentation and accumulation technique,which remarkedly reduces matrix-induced interferences and effectively reduces space charge effects.Serum samples were pretreated with methanol to precipitate proteins and the supernatants were measured by QLIT-6610MD spectrometer.The linear correlation coefficient(R^(2)>0.9990)is obtained for MTX in the range of 10-5000μg/L.The limit of quantification is 10μg/L.The precision of the quality control is better than 6.94%,and the recoveries of spiked serum samples are 86.39%-97.84%with a coefficient of variation less than 8.04%.We used the QLIT-6610MD and AB QTRAP 6500+liquid chromatography-mass spectrometers to analyze 70 clinical serum samples for comparison of the performance of the house-made instrument by means of Pearson’s coefficient and BLand-ALtman method.The results showed that the QLIT-6610MD mass spectrometry achieves a similar level of performance to AB ATRAP 6500+instrument,and provides a new domestic instrument option for clinical mass spectrometry.In addition,this study explores the possibility applying the multi-stage fragmentation technique(MS^(n))to determine MTX in serum,gaining fundamental data for future studies.