Pitfalls in Cefepime Titration from Human Plasma: Plasma- and Temperature-Related Drug Degradation In Vitro

Abstract

While developing a high-pressure liquid chromatography assay for cefepime in plasma, we observed significant drug degradation at 20 and 37°C but not at 4°C. This plasma-related degradation persisted after protein removal. This warrants caution regarding cefepime assays for pharmacokinetic and pharmacodynamic studies of cefepime in vitro and in vivo.While a high-pressure liquid chromatography (HPLC) method to measure levels of cefepime in plasma was being validated, significant plasma-dependent and temperature-dependent degradation of cefepime was observed. Since this might bias cefepime determination in clinical samples, the degradation process was further explored. Experiments were performed in the presence or absence of plasma and at temperatures of 4, 20, and 37°C. In the presence of plasma, maintaining the temperature at 4°C was critical to avoid degradation. At higher temperatures, conforming either to bench-top storage (20°C) or incubation at physiological temperature (37°C), rapid degradation occurred.Methods and experimental design. Cefepime was obtained from Bristol-Myers Squibb AG (Sermoneta, Italy). Deionized water (Ͼ16 M⍀ ⅐ cm) was used. Healthy volunteers' plasma (stored at Ϫ30°C) was obtained from 450-ml individual blood collection bags (Fenwal, Baxter, Volketswil, Switzerland) containing 63 ml of a solution of citric acid (15.6 mM), sodium citrate (90 mM), sodium dihydrogenophosphate (16.1 mM), dextrose (160 mM), and adenine (2 mM).All samples were initially prepared at 4°C. Before HPLC determination, plasma was deproteinized either by precipitation followed by back extraction or by ultrafiltration. For precipitation, 500 l of plasma was mixed with 500 l of acetonitrile, allowed to stand for 10 min, and centrifuged at 15,000 ϫ g for another 10 min. For back extraction, 500 l of the deproteinized supernatant was mixed with 1 ml of methylene chloride, allowed to stand for 10 min, and centrifuged as described above. The supernatant was injected into the HPLC. Ultrafiltration was performed as described elsewhere (10).Cefepime was separated on a C 18 column (LC 18 , 15 cm by 4.6 mm; Supelco, Bellefonte, Pa.), using a Merck-Hitachi HPLC LaChrom system (Hitachi Instruments, Ichige Hitachinaka, Japan) with a diode array detection. The mobile phase (1 ml/min) comprised 92% volume of 0.2 M borate adjusted to pH 6.8 (20°C) with NaOH and 8% volume of methanol. Samples were sealed to prevent evaporation and kept at a controlled temperature (Peltier cooler; Labsource, Reinach, Switzerland) until their automated injection (50 l). Cefepime was quantified by computing the area under the peak from the 260-nm absorption signal. Calibration curves, consisting of seven standards (0.5 to 200 mg/liter) were prepared daily and assayed both at the beginning and the end of the procedure. Linearity, extraction recovery, intrarun and interrun coefficients of variation and accuracy, limit of detection (LOD), and limit of quantification (LOQ) were determined (14).Assay validation. The basic performance of the method was excell...

Determination of ceftazidime and cefepime in plasma and dialysate-ultrafiltrate from patients undergoing continuous veno-venous hemodiafiltration by HPLC

“…Cherti et al [25] and Elkhaïlï et al [26] described important degradation of aqueous extracts originating from plasma after sample pre-treatment in cefepime. Bugnon et al [28] also showed that cefepime degradation in plasma samples at room temperature occurred in spite of prior plasma deproteinization and recommended to maintain samples at 4 • C. The stability of samples used for pharmacokinetic analyses is guaranteed with our HPLC method for the period of time described above.…”

“…Although other HPLC methods have been published in the literature for ceftazidime [20][21][22][23] and cefepime [24][25][26][27] determination in different fluids, only a few of the works mentioned above provide data about drugs' stability [22,25,26,28]. Our work includes a complete stability study because stability assessment is considered a fundamental parameter for the validation of bioanalytical methods [16,29], and is critical for proper interpretation of analytical results [13].…”

Monitoring of cefepime in human serum and plasma by micellar electrokinetic capillary chromatography: Improvement of sample preparation and validation by liquid chromatography coupled to mass spectrometry

“…Data gathered thus far suggest that TDM can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis . Analytical methods for monitoring cefepime in biosamples include bioassays , HPLC with UV detection , LC–MS , and MEKC . They should be capable of accurately monitoring cefepime levels in the range of 1 to 50 μg/mL .…”

High Cefepime Plasma Concentrations and Neurological Toxicity in Febrile Neutropenic Patients with Mild Impairment of Renal Function

“…Tubes were centrifuged at 4°C within 1 h and stored at Ϫ80°C until analysis. Cefepime plasma concentrations were measured by a UV high-performance liquid chromatography method validated according to international guidelines (5). The analytical range was 0.25 to 200 mg/liter, and the intrarun/interrun accuracy and coefficient of variation were Ͼ95% and Ͻ10%, respectively.…”