A sensitive method for analyzing fluconazole in extremely small volumes of neonatal serum

Abstract

Background: The need for a large volume of serum sample significantly reduces the feasibility of neonatal pharmacokinetic studies in daily practice, which must often rely on scavenged or opportunistic sampling. This problem is most apparent in preterm newborns, where ethical and practical considerations prohibit the collection of large sample volumes. Most of the fluconazole analysis assays published thus far required a minimum serum sample of 50 to 100 μL for a single assay. The purpose of the present study was to develop and validate a sensitive method requiring a smaller sample volume (10 μL) to satisfy clinically relevant research requirements. Methods: Following simple protein precipitation and centrifugation, the filtrated supernatant was injected into a liquid chromatography system and separated with a C18 reverse-phase column. Fluconazole and the internal standard (IS, fluconazole-d4) were detected and quantified using tandem mass spectrometry. The method was validated with reference to the Food and Drug Administration's Guidance for Industry. Accuracy and precision were evaluated at six quality control concentration levels (ranging from 0.01 to 100 μg/mL). Results: Investigated calibration curves were linear in the 0.01-100 μg/mL range. Intra-and inter-day accuracy (− 7.7 to 7.4%) and precision (0.3 to 6.0%) were below 15%. The calculated limit of detection and the lower limit of quantification (LLOQ) was 0.0019 μg/mL and 0.0031 μg/mL, respectively. Fluconazole in the prepared samples was stable for at least 4 months at − 20°C and − 80°C. This method was applied to analyze 234 serum samples from ten neonates who received fosfluconazole, a water-soluble phosphate prodrug of fluconazole which converts to fluconazole in the body, as part of a pharmacokinetic study using daily scavenged laboratory samples. The median (range) concentration up to 72 h after fosfluconazole administration was 2.9 (0.02 to 26.8 μg/mL) μg/mL, which was within the range of the calibration curve. Conclusion: Fluconazole was able to be detected in an extremely small volume (10 μL) of serum from neonates receiving fosfluconazole. The method presented here can be used to quantify fluconazole concentrations for pharmacokinetic studies of the neonatal population by using scavenged samples.

Review of Scavenged Sampling for Sustainable Therapeutic Drug Monitoring: Do More With Less

“…Eventually, 19 of the 23 studies were included; 4 studies that did not meet the inclusion criteria were used to support the concept of scavenged sampling. 8,9,11–31 Table 1 presents the characteristics of the 19 included studies. Table 2 presents the outcomes and limitations of included studies as described by the authors.…”

Fluconazole Population Pharmacokinetics after Fosfluconazole Administration and Dosing Optimization in Extremely Low-Birth-Weight Infants

“…The total serum FLCZ concentration was measured using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (TSQ Vantage LC-MS with Dionex UltiMate 3000 RSLC system; Thermo Fisher Scientific K.K., Tokyo, Japan) in the hospital’s internal laboratory using a validated method ( 28 ). FLCZ (>99.0%) and its internal standard (IS; FLCZ-d4 stable isotope [>99.0%]) were purchased from Toronto Research Chemical (North York, ON, Canada).…”

Pharmacokinetics of Fluconazole tablets administered to healthy subjects ‎

“…High-performance liquid chromatography coupled with UV detector was used for measuring fluconazole's plasma concentrations applying analytical methods described previously with phenacetin as the internal standard at 210 nm [32][33][34][35][36]. The analytical method was validated applying the current recommended FDA bioanalytical method validation guidelines [37][38][39][40].…”

“…The analytical method applied in this investigation [32][33][34][35][36] was successfully applied to demonstrate the pharmacokinetics after a single dose of 200 mg fluconazole tablets since all the validation parameters including accuracy, specificity, selectivity, sensitivity, and precision were well within the acceptance ranges required by the current recommended FDA bioanalytical method validation guidance [37][38][39][40]. Moreover, fluconazole's plasma concentration ranges from 50-10000 ng/ml applied were quite enough for covering the resulted plasma concentrations ranges as depicted in Figure 2.…”