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-1 for 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-1 and 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.

CNS pharmacokinetics of antifungal agents.

The goal in treatment of infections is to achieve a beneficial effect while minimizing toxicity. It is widely recognized that the principles of pharmacokinetics and pharmacodynamics are critical to determining an adequate dose-response relationship. There has been an increased involvement of the CNS to infection from opportunistic and endemic fungi over the last several decades due to establishment of solid-organ and bone marrow transplantation as well as immunosuppression from HIV. In this regard it has become critical to define optimal dosing regimens by an understanding of the processes which govern delivery of an antifungal agent to the targeted CNS site of involvement. The objective of this review is to: i) summarize published experimental and clinical antifungal pharmacokinetics; and ii) examine the relationship between CNS antifungal pharmacokinetics and efficacy. Examination of these studies reveal marked variability among antifungal drugs with regard to cerebrospinal fluid and brain parenchymal penetration. Formal examination of the relationship between CNS antifungal pharmacokinetics and efficacy are limited. The few experimental studies available suggest that brain parenchymal kinetics is a superior predictor of antifungal efficacy than cerebrospinal fluid concentrations.

Treatment of experimental cryptococcal meningitis with fluconazole: impact of dose and addition of flucytosine on mycologic and pathophysiologic outcome.

Fluconazole is effective in the therapy of cryptococcal meningitis in patients with AIDS. The optimal dosage of fluconazole and the impact of combination with flucytosine are not known. In this study, rabbits with experimental cryptococcal meningitis were given fluconazole at low, intermediate, or high dose or in combination with a low or intermediate dose of flucytosine. Serial cerebrospinal fluid (CSF) examinations showed that all three doses of fluconazole and low-dose fluconazole in combination with intermediate-dose flucytosine were effective in reducing CSF cryptococcal titer, lactate, white blood cell count, and cryptococcal antigen (CRAG) titers. The intermediate and high doses of fluconazole reduced CSF fungal (P < .05) and CRAG (P < .001) titers earlier than low-dose fluconazole alone or in combination with flucytosine. Only the highest dose of fluconazole reduced brain edema after 7 days. In this model of cryptococcal meningitis, there was evidence of a dose response with fluconazole but no in vivo synergism with flucytosine.

Systemic and disseminated candidiasis complicating acute renal failure.

One case of disseminated Candida albicans infection and two cases of systemic C. albicans infection in patients with acute renal failure are described. The predisposing factors and management implications are discussed and therapeutic recommendations made.

Liquid-chromatographic determination of 5-fluorocytosine.

We report a "high-performance" liquid-chromatographic method for measuring 5-fluorocytosine in plasma and cerebrospinal fluid. After deproteinization with trichloroacetic acid, the supernates are chromatographed on a reversed-phase (C18) column. Response to concentration is linear in the range of 5 to 200 mg/L, with ultraviolet detection at 276 nm. The assay requires only 0.1 mL of plasma, is reproducible, and may be performed in less than 12 min. 5-Fluorocytosine concentrations determined by this procedure correlated well with those obtained by spectrofluorometry, although the present method is more specific with no observable interference from co-administered amphotericin B and most other commonly encountered drugs, including salicylat:. This method is applicable to the routine therapeutic monitoring of pediatric and adult patients as well as to pharmacokinetic studies.

Pharmacological studies with 5-fluorocytosine.

A cylinder plate bioassay for 5-fluorocytosine (5-FC) is described which permits determination of 5-FC concentrations in biological fluids in the presence of amphotericin B. Using this assay, we determined serum concentrations in 12 patients after a single dose of drug and in 8 patients receiving daily 5-FC at 6-hr intervals (4 to 120 days). Drug was detectable in serum as early as 0.5 hr and concentrations were measurable as long as 24 hr after a dose, regardless of renal function. Peak concentrations occurred 6 hr after the initial drug dose, but were seen between 1 and 2 hr after a dose in all patients receiving a minimum of 4 days of therapy. Mild to moderate renal impairment produced marked increases in peak 5-FC concentrations in the serum of a group of eight patients on three different dosage schedules. Five patients were studied before and after amphotericin B-induced renal insufficiency. Peak concentrations increased from 14 to 142% concomitant with the change in renal function. Parallel studies in rabbits confirmed the results in our patients. 5-FC half-life in the serum of 10 rabbits increased from 3.35 +/- 0.27 to 24.63 +/- 0.70 hr after experimentally induced acute renal failure. Concentrations of 5-FC in the cerebrospinal fluid of five patients ranged from 17 to 62 mug/ml and were 74.4 +/- 5.6% of simultaneously determined serum concentrations. The effect of renal function on 5-FC concentrations in cerebrospinal fluid was similar to that seen with serum.