Center of Basic Molecular Science (CBMS), Department of Chemistry, Tsinghua University, Beijing 100084, PR China.
Infectious Diseases Dependent, Tianjin Second People's Hospital, Tianjin 300192, PR China.
Talanta. 2017 Apr 1;165:449-457. doi: 10.1016/j.talanta.2016.12.052. Epub 2016 Dec 30.
Amphotericin B (AMB), fluconazole (FZ), and fluorocytosine (FC) are recommended for HIV-associated cryptococcal meningitis (CM) patients as preferred antibiotics. This study presents a fast and automated online-dual-solid phase extraction (SPE)-LC coupled with high resolution mass spectrometer (HRMS) method to simultaneously measure the concentrations of AMB, FZ, and FC in human plasma and cerebrospinal fluid (CSF). Automated sample clean-up was performed on the human plasma and CSF samples with stop-flow heart-cutting two dimensional (2D) separation using a online-dual-SPE system, allowing retention and accumulation of AMB, FZ, and carbamazepine (CBZ, Internal standard (IS)) by the OasisHLB cartridge, and retention and accumulation of FC and 5-methylcytosine hydrochloride (MC, IS) by the HyperSep Hypercarb cartridge respectively. Followed by LC elution, quantification by Q-Exactive Hybrid Quadrupole-Orbitrap with targeted-selected ion monitoring (t-SIM) mode was applied to simultaneously determine the concentrations of AMB, FZ and FC. The bioanalysis was achieved in a total running time of 7min. The method was fully validated according to FDA guidelines. The lowest limit of quantification (LLOQ) was 0.04, 0.04, and 0.40μgmL for AMB, FZ, and FC, respectively. AMB, FZ, and FC levels were linear in the ranges of 0.04-2.00μgmL, 0.04-2.00μgmL and 0.40-20.00μgmL, respectively. The method showed good performance for human plasma and CSF samples with linearity (R>0.99), intra-day and inter-day precision (relative standard deviation, RSD<4.32% and <4.06%, respectively), recovery (89.93-93.28% and 90.09-93.58%, respectively) and matrix effect (96.35-103.78% and 92.32-101.48%, respectively). The validated method was successfully applied in real samples of Chinese patients. Overall, our results indicate that this fully automated, sensitive, and reliable online-dual-SPE-LC-HRMS method is effective for therapeutic drug monitoring (TDM) of AMB, FZ, and FC levels.
两性霉素 B（AMB）、氟康唑（FZ）和氟胞嘧啶（FC）被推荐用于 HIV 相关隐球菌性脑膜炎（CM）患者作为首选抗生素。本研究提出了一种快速、自动化的在线双固相萃取（SPE）-LC 与高分辨率质谱（HRMS）联用方法，可同时测量人血浆和脑脊液（CSF）中 AMB、FZ 和 FC 的浓度。使用在线双 SPE 系统进行自动样品净化，通过停流心切二维（2D）分离，使 AMB、FZ 和卡马西平（CBZ，内标（IS））保留和积累在 OasisHLB 卡上，FC 和 5-甲基胞嘧啶盐酸盐（MC，IS）保留和积累在 HyperSep Hypercarb 卡上。然后进行 LC 洗脱，采用靶向选择离子监测（t-SIM）模式的 Q-Exactive Hybrid Quadrupole-Orbitrap 进行定量分析，可同时测定 AMB、FZ 和 FC 的浓度。总运行时间为 7min。该方法完全按照 FDA 指南进行验证。AMB、FZ 和 FC 的最低定量下限（LLOQ）分别为 0.04、0.04 和 0.40μgmL。AMB、FZ 和 FC 的线性范围分别为 0.04-2.00μgmL、0.04-2.00μgmL 和 0.40-20.00μgmL。该方法对人血浆和 CSF 样品具有良好的性能，线性度（R>0.99）、日内和日间精密度（相对标准偏差，RSD<4.32%和<4.06%）、回收率（89.93-93.28%和 90.09-93.58%）和基质效应（96.35-103.78%和 92.32-101.48%）。验证后的方法成功应用于中国患者的实际样本。总的来说，我们的结果表明，这种全自动、灵敏、可靠的在线双 SPE-LC-HRMS 方法可有效用于 AMB、FZ 和 FC 水平的治疗药物监测（TDM）。
