UPLC-MS/MS法同时测定人血浆和脑脊液中3种抗神经精神性狼疮药物的浓度

Simultaneous determination of three anti-NPSLE drugs in human plasma and cerebrospinal fluid by UPLC-MS/MS

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 develop a UPLC-MS/MS method for simultaneous determination of three anti-NPSLE drugs methylprednisolone （ MP ）, cyclophnosphamide （ CP ） and methotrexate （ MTX ） in human plasma and cerebrospinal fluid （ CSF ）, and evaluate their blood-brain barrier penetration rate in NPSLE patients. Method： With 4-aminoacephenone as internal standard （ IS ）, the plasma and CSF samples were treated by protein precipitation and the UPLC-MS/MS method was used to detect the substances simultaneously. The chromatographic conditions： using a Waters ACQUITY UPLC CSH column （2.1 mm × 50 mm, 1.7 μ m ）in an aqueous solution containing 0.1% formic acid and acetonitrile containing 0.1% formic acid as a mobile phase at a flow rate of 0.4 mL· min-1, and the column temperature was 30 ℃ ; The mass spectrometry conditions： electrospmy ionization （ ESI ）, positive ion and negative ion scanning, and multi reaction monitoring （ MRM ） scanning for mass spectrometry. MP was a negative ion scan （ m/z 373.1 → 343.2 ） ; CP and MTX were positive ion scans （ m/z 261 → 233, m/z 455 → 308 ） ; internal standard 4-aminoacetophenone was a positive ion scan （ m/z 136 →93.8 ）. In this study, we investigated the stability of the autosampler placement and dilution reliability after the preparation of three compounds in plasma samples; and CSF samples were prepared at room temperature, the placement stability and dilution reliability of the automatic injector. Results： The standard curves concentration ranges of the three drugs in plasma were MP 0.5-500 ng·mL-1, CP 0.5-200 ng· mL-1, MTX 0.5-500 ng· mL-1, respectively; and the standard curves concentration range of the three drugs in cerebrospinal fluid was 0.5 - 200 ng· mL-1. The intra-day and inter-day precisions （ RSD ） of the compounds in plasma and cerebrospinal fluid were〈15%, and the relative errors were within the range of± 15%. Matrix effect, extraction recovery and stability were also validated respectively. The three compounds remained stable during sample storage and handling progress. The method was used to detect the plasma and corresponding time CSF samples from NPSLE patients treated with MP, CP and MTX to evaluate the blood-brain barrier penetration of the three drags. Conclusion： This method is simple, sensitive and accurate, and is suitable for the research of blood-brain barrier penetration rate of three anti-NPSLE drugs. And the rationality of the clinical protocol was verified in the application of this method.