Utilizing online-dual-SPE-LC with HRMS for the simultaneous quantification of amphotericin B, fluconazole, and fluorocytosine in human plasma and cerebrospinal fluid.
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 Oasis®HLB 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-1for AMB, FZ, and FC, respectively. AMB, FZ, and FC levels were linear in the ranges of 0.04-2.00μgmL-1, 0.04-2.00μgmL-1and 0.40-20.00μgmL-1, respectively. The method showed good performance for human plasma and CSF samples with linearity (R2>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.
Center of Basic Molecular Science (CBMS), Department of Chemistry, Tsinghua University, Beijing 100084, PR China.
Lihua
L
Qu
Infectious Diseases Dependent, Tianjin Second People's Hospital, Tianjin 300192, PR China.
Jing
J
Qian
Infectious Diseases Dependent, Tianjin Second People's Hospital, Tianjin 300192, PR China. Electronic address: mapingtianjin@163.com.
Ping
P
Ma
Center of Basic Molecular Science (CBMS), Department of Chemistry, Tsinghua University, Beijing 100084, PR China. Electronic address: yinzheng@mail.tsinghua.edu.cn.
Zheng
Z
Yin
D000666:Amphotericin B / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D000935:Antifungal Agents / Q000032:analysis / Q000097:blood / Q000134:cerebrospinal fluid; D002853:Chromatography, Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D015725:Fluconazole / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D005437:Flucytosine / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D006801:Humans; D009862:Online Systems; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000935:Antifungal Agents; D000666:Amphotericin B; D015725:Fluconazole; D005437:Flucytosine
HIV-CM; Human plasma and CSF; Online-dual-SPE-LC-HRMS; Stop-flow heart-cutting 2D