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目的:建立同时检测人血浆、脑脊液中甲泼尼龙(methylprednisolone,MP)、环磷酰胺(cyclophnosphamide,CP)和甲胺喋呤(methotrexate,MTX)的UPLC-MS/MS法,初步评价了3种药物在神经精神性狼疮(NPSLE)患者中的血脑屏障穿透率。方法:以4-氨基苯乙酮为内标物,对血浆及脑脊液样品进行蛋白沉淀处理后,采用UPLC-MS/MS法同时检测各物质。色谱条件:采用ACQUITY UPLC CSH色谱柱(2.1 mm×50 mm,1.7μm),以含0.1%甲酸的水溶液及含0.1%甲酸的乙腈为流动相,梯度洗脱,流速0.4 m L·min~(-1),柱温30℃;质谱条件:电喷雾离子源(ESI),正离子和负离子扫描,采用多反应监测(MRM)的质谱扫描方式进行测定。MP为负离子扫描(m/z 373.1→343.2);CP及MTX为正离子扫描(m/z261→233,m/z 455→308);内标4-氨基苯乙酮为正离子扫描(m/z 136.0→93.8)。本实验考察了3个化合物血浆样品制备后自动进样器放置的稳定性和稀释可靠性;以及脑脊液样品室温放置、制备后自动进样器放置稳定性和稀释可靠性。结果:血浆中3种药物标准曲线浓度范围分别为MP 0.5~500 ng·m L~(-1),CP0.5~200 ng·m L~(-1),MTX 0.5~500 ng·m L~(-1);脑脊液中3个药物标准曲线浓度范围均为0.5~200 ng·m L~(-1)。血浆和脑脊液中化合物的批内、批间精密度(RSD)均<15%,相对误差均在±15%范围内。基质效应、萃取回收率和稳定性也分别进行了验证。且样本储存和处理过程中,3个化合物能够保持稳定。本方法用于检测使用MP,CP和MTX治疗的NPSLE患者的血浆与相应时间的脑脊液样本,用于评价3种药物的血脑屏障穿透率。结论:本方法简便、灵敏、准确,适用于3种药物的血脑屏障穿透率研究,且在方法应用中验证了临床方案的合理性。
OBJECTIVE: To establish a UPLC-MS / MS method for the simultaneous detection of methylprednisolone (MP), cyclophnosphamide (CP) and methotrexate (MTX) in human plasma and cerebrospinal fluid Blood-brain barrier penetration rate of drug in patients with neuropathic lupus (NPSLE). METHODS: Plasma and cerebrospinal fluid samples were subjected to protein precipitation with 4-aminoacetophenone as an internal standard. The substances were simultaneously detected by UPLC-MS / MS. Chromatographic conditions: The mobile phase was eluted with ACQUITY UPLC CSH column (2.1 mm × 50 mm, 1.7 μm) with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile with a flow rate of 0.4 mL · min -1 -1) with a column temperature of 30 ℃. Mass spectrometry conditions were electrospray ionization (ESI), positive and negative ion scanning, and the mass spectrometry scan was performed using multiple reaction monitoring (MRM). MP was negative ion scan (m / z 373.1 → 343.2); CP and MTX positive ion scan (m / z 261 → 233, m / z 455 → 308); internal standard 4-aminoacetophenone positive ion scan z 136.0 → 93.8). In this experiment, we investigated the stability and dilution reliability of the autosampler after the preparation of three plasma samples. The CSF samples were placed at room temperature, and the autosampler was placed on the stability and dilution reliability after preparation. Results: The concentration range of standard curve of three drugs in plasma ranged from 0.5 to 500 ng · m L (-1) for MP, 0.5 to 200 ng · m L (-1) for CP and 0.5 to 500 ng · m L for CPX ~ (-1). The standard curve concentrations of three drugs in CSF ranged from 0.5 to 200 ng · m L -1. The intra- and inter-assay precision (RSD) of compounds in plasma and cerebrospinal fluid were both <15% with relative errors within ± 15%. Matrix effects, extraction recovery and stability were also validated separately. Three compounds remained stable during sample storage and handling. This method was used to detect plasma and time-appropriate cerebrospinal fluid samples of NPSLE patients treated with MP, CP and MTX for the evaluation of blood-brain barrier penetration of the 3 drugs. Conclusion: The method is simple, sensitive and accurate, suitable for the study of blood-brain barrier permeability of three kinds of drugs, and the rationality of the clinical scheme is verified in the application of the method.