•A quantitative strategy for plasma and cerebrospinal fluid samples was developed.•The method was based on online-dual-SPE-LC-HRMS.•Automated SPE clean-up was based on two dimensional (2D) technique.•The method has been applied at clinics in TDM of four Chinese HIV-CM patients.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 7 min. The method was fully validated according to FDA guidelines. The lowest limit of quantification (LLOQ) was 0.04, 0.04, and 0.40 μg mL−1 for AMB, FZ, and FC, respectively. AMB, FZ, and FC levels were linear in the ranges of 0.04–2.00 μg mL−1, 0.04–2.00 μg mL−1 and 0.40–20.00 μg mL−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.A quantitative strategy of amphotericin B, fluconazole, and fluorocytosine in human plasma and cerebrospinal fluid was developed with online-dual-SPE-LC-HRMS using t-SIM mode.

•A quantitative strategy for the analysis of antiepileptic drugs was developed.•The method was based on online-SPE-LC-HRMS/MS.•The method was validated following the FDA guidelines.•The method has been applied at clinics in TDM of four Chinese epilepsy patients.A simple and efficient bioanalytical method for simultaneous determination of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), and its active metabolite carbamazepine 10,11-epoxide (CBZE) in human plasma using online solid phase extraction (SPE)-liquid chromatography (LC) coupled with high resolution mass spectrum (HRMS) under targeted MS/MS (t-MS2) analysis mode has been developed. The procedure integrated an automated sample clean-up of human plasma by Oasis®HLB SPE cartridge, a separation by ZORBAX SB-C18 analysis column, and a quantification by Q-Exactive Hybrid Quadrupole-Orbitrap. The total running time was 13 min. The lower limit of quantification (LLOQ) of PB, PHT, CBZ, and CBZE were 0.008, 0.008, 0.0016 and 0.0016 μg mL−1 respectively and the linearities were in the range of 0.008–2.500, 0.008–2.500, 0.0016–0.500 and 0.0016–0.500 μg mL−1 respectively. The mean recovery was between 91.82% and 108.27% and the matrix effect was between 93.29% and 102.09%. The relative standard deviations of interday and intraday were less than 6.41%. The method has been successfully applied in therapeutic drug monitoring (TDM) of four Chinese epilepsy patients. This fully automated, simple, sensitive and reliable online-SPE-LC-HRMS/MS method serves well for TDM of PB, PHT, CBZ and CBZE at clinics for either single or combination treatment.A quantitative strategy of PB, PHT, CBZ and CBZE in human plasma was developed with online-SPE-LC-HRMS/MS using t-MS2 mode.

A quantitative strategy for the analysis of sofosbuvir (SF) in human plasma has been developed with an online solid phase extraction (SPE)-liquid chromatography (LC) coupled with high resolution mass spectrometry (HRMS) system using a MS/MS targeted ion fragmentation scan (t-MS2), targeted-selected ion monitoring (t-SIM) and full MS-SIM (F-SIM) mode. The sample was pretreated automatedly and analyzed within 10 min via an online SPE system equipped with an Oasis®HLB (2.1 × 20 mm, 5 μm) SPE column and LC system with a ZORBAX SB-C18 (4.6 × 250, 5 μm) analytical column. A Q-Exactive hybrid quadrupole-Orbitrap HRMS was utilized for positive identification and quantification. Method detection limits ranged between 0.5 and 2000 ng mL−1 in human plasma for both t-MS2 and t-SIM mode and between 2 and 2000 ng mL−1 for F-SIM mode. In all three modes, the overall intra-day and the inter-day variations were less than 8.07%. The recovery of all three methods was in the range of 92.06–107.20% with RSD% less than 3.90%. The matrix effect of all three methods was in the range of 94.82–101.89% with RSD% less than 7.69%. The optimized two methods (t-MS2 and t-SIM) demonstrated good performance in terms of specificity, lower limit of quantification (LLOQ), linearity, recovery, precision and accuracy.