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Abstract
Introduction Cefiderocol is a new siderophore time-dependent antibiotic of last resort. The manufacturer reports a stability of 6 hours for the infusion solution diluted in normal saline (NS) or dextrose 5% in water (D5W) for a concentration between 7.5 and 20 mg/mL. Optimising its effectiveness by continuous infusion is crucial. The aim of this work was to study the physicochemical stability of cefiderocol diluted in NS or D5W in polypropylene syringes for 48 hours at a concentration of 62.5 mg/mL stored at room temperature, protected or not from light.
Materials and methods Three preparations for each condition were performed. At each time of the analysis, one sample for each preparation was analysed in triplicate by a validated high performance liquid chromatography method coupled to a photodiode array detector at 260 nm. Particle contamination, absorbance measurement, visual inspection and pH measurement were assessed. The limit of stability was set at 90% of the initial concentration, without physical modification.
Results The linearity was validated with an R² of 0.9999. The coefficients of variation for repeatability and intermediate precision were less than 2%. In NS and D5W, cefiderocol retained more than 90% of the initial concentration after 12 hours in syringes, exposed or not to light. Two degradation products (nos 2 and 11, observed during forced degradation) were detected during the stability study. The absorbance at 410 nm increased progressively, regardless of the storage conditions. The particulate contamination test met the specifications of the container. pH values were all between 5.22 and 5.32. No visual changes were detected.
Conclusion In polypropylene syringes, cefiderocol 62.5 mg/mL (3 g in 48 mL) diluted in NS or D5W was stable for 12 hours at room temperature. These new data allow the use of cefiderocol in continuous infusion.
- critical care
- administration
- intravenous
- pharmaceutical preparations
- microbiology
- microbiology
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
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INTRODUCTION
Cefiderocol is a new cephalosporin approved by the European Medicines Agency in 2020.1 It is used as an antibiotic of last resort to treat an infection caused by a carbapenemase-producing Gram-negative bacterium. The spectrum also covers Pseudomonas aeruginosa and non-fermentative bacteria such as Acinetobacter baumanii and Stenotrophomonas maltophilia. Cefiderocol has the ability to bind to free extracellular iron via the siderophore on its side chain (figure 1), allowing active transport into the periplasmic space of Gram-negative bacteria through iron chelation systems.2 Cefiderocol then binds to penicillin-binding protein. It is the siderophore group that protects cefiderocol from carbapenemases such as oxacillin carbapenemase, New Delhi metallo-β-lactamase and Verona integron-encoded metallo-β-lactamase.3 4
Structure–activity relationships for cefiderocol.
The recommended dosage for treating infections is 6 g per day, divided into three injections, with an infusion time of 3 hours. The manufacturer reports a stability of 6 hours for the infusion solution (diluted in normal saline (NS) or dextrose 5% in water (D5W)) for a concentration between 7.5 and 20 mg/mL and stored at room temperature.5
Patients in intensive care units (ICUs) are a population at risk. For pathophysiological reasons, most patients have fluid restriction, which does not allow high-volume infusions.6–8 Drugs are administered in small volumes and in flow-controlled syringes. However, administration in a small volume leads to very concentrated solutions—for example, for cefiderocol a concentration of 62.5 mg/mL is obtained (3 g diluted in 48 mL). To the best of our knowledge, no stability studies have been performed at such high concentrations.
Cefiderocol belongs to the β-lactam class and is therefore a time-dependent antibiotic. The aim of the treatment is to rapidly reach a concentration of 4 times the minimum inhibitory concentration and to maintain this concentration for the duration of the treatment period.9 Optimising its effectiveness by continuous infusion is therefore crucial.10 However, this approach needs to be validated with a stability study.
The aim of this work was to study the physicochemical stability of cefiderocol diluted in NS or D5W in polypropylene syringes for 48 hours at a concentration of 62.5 mg/mL stored at room temperature, protected or not from light, in order to simulate the administration by infusion in an ICU.
Materials and method
Chemicals and reagents
Potassium dihydrogen phosphate (KH2PO4) (Merck; batch: AM1089577747), methanol for high performance liquid chromatography (HPLC) (Carlo Erba; batch: V0D04260D) and orthophosphoric acid (VWR; batch 17B174014) were used for the mobile phase. Hydrochloric acid 1 M (VWR Chemicals; batch: 200618C011), sodium hydroxide 1 M (VWR Chemicals; batch: 200416C004) and hydrogen peroxide 30% (Merck; batch: K48743810713) were used for the forced degradation. Cefiderocol (Fetcroja) 1 g, powder for injection, for intravenous use (Shionogi; batch: FEFR0120), NS 250 mL glass vial (Chaix et du Marais, Lavoisier, batch: 9F582) and D5W 250 mL glass vial (Chaix et du Marais, Lavoisier, batch: 9F591) were used for preparation of syringes. Water for injection 500 mL (Chaix et du Marais, Lavoisier, batch: 0F564) and Water for chromatography (obtained from a reverse osmosis system) (Millipore Iberica, Madrid, Spain) were used for test solutions and dilution. Toluene-sulfonic acid sodium for synthesis (Merck; batch: S7975925108); sucrose 24% (Algopedol) (PEDCO Pharma; batch: 04220) were used to assess the specificity.
Preparation of test solutions
All manipulations were performed under a biological safety cabinet. As recommended by the summary of product characteristics, cefiderocol should be reconstituted with 10 mL NS or D5W to obtain a 89.3 mg/mL solution of cefiderocol (11.2 mL displacement volume).5 For cost reasons, a single vial was reconstituted and then filled with the same solvent to a volume of 16 mL in a polypropylene syringe (Omnifix, B Braun; 20 mL; batch: 20M09C8) to obtain a concentration of 62.5 mg/mL. This allows simulation of a 48 mL syringe at 62.5 mg/mL (real condition) without using three vials.
Chemical stability
HPLC assay
Cefiderocol solutions were analysed by stability-indicating reversed-phase HPLC (RP-HPLC) with photodiode array detection, adapted from a method developed in our laboratory for the determination of ceftazidime/avibactam. The HPLC system consisted of an ELITE LaChrom VWR/Hitachi plus autosampler, a VWR photodiode array (PDA) detector L-2455 and a VWR L-2130 HPLC pump. The column used was LiChrospher 100 RP-18, LiChroCART 125–4, length 12.5 cm and particle size 5 µm (Analytical Chromatography, Merck). The photodiode array detector evaluates the UV spectrum of the chromatographic column effluent every 0.4 s. The wavelength of the analysis spectrum was between 190 and 400 nm. Data acquisition and peak interpretation was performed with the EZChrome Elite software (VWR, Agilent). The mobile phase consisted of 0.05 mol/L buffer KH2PO4 (A) and methanol (B). The pH of the mobile phase was adjusted to pH 3 with orthophosphoric acid. This was a gradient method: the gradient starts with a ratio of 83/17 (A/B), then linearly changes to 70/30 at T7 min and is maintained until T15 min, changes again to 83/17 at T17 min and is maintained until the end of the analysis at T20 min. The flow rate was set at 1.5 mL/min, with an injection volume of 50 µL. The detection wavelength was set at 260 nm. The temperature of the injector was set at 5°C and the temperature of the column oven at 30°C.
Validation method
The calibration curve was constructed from the plots of peak area versus concentration obtained from the manufacturer’s powder. The linearity of the method was evaluated with five concentrations (25, 37.5, 50, 62.5 and 75 µg/mL) of cefiderocol, diluted in ultrapure water. For the calibration curves, the homogeneity of the variances was evaluated with a Cochran test whose significance level was set at p<0.05. An analysis of variance (ANOVA) of the linear regression data was performed to assess the significance (p<0.05) of the proposed method. The intra-day repeatability was evaluated as recommended by the International Conference on Harmonisation (ICH) Q2 (R1)11 using three determinations for each concentration at 25, 50 and 75 µg/mL. For inter-day precision, three determinations for each concentration at 25, 50 and 75 µg/mL of cefiderocol solutions were assayed daily on three different days.
Forced degradation and specificity
The stability-indicating capability was evaluated by analysing forced degraded cefiderocol solutions. The aim of this forced degradation was to obtain degradation products with retention times different from those of cefiderocol. The objective was to obtain a degradation close to 20% of our molecule of interest.12 Conditions for the degradation of cefiderocol were as follows:
Acidic condition: 1 mL of a 200 µg/mL cefiderocol solution was diluted with 1 mL HCl 1 M, stored at room temperature for 15 hours, neutralised by 1 mL NaOH 1 M and diluted with 1 mL ultrapure water to obtain a theoretical concentration of 50 µg/mL.
Alkaline condition: 1 mL of a 200 µg/mL cefiderocol solution was diluted with 1 mL NaOH 0.01 M, stored at 20–25°C for 5 min, neutralised by 1 mL HCl 0.01 M and diluted with 1 mL ultrapure water to obtain a theoretical concentration of 50 µg/mL.
Oxidative degradation: 1 mL of a 200 µg/mL cefiderocol solution was diluted with H2O2 0.3% 1 mL, stored at 35°C for 1 hour and diluted with 2 mL ultrapure water to obtain a theoretical concentration of 200 µg/mL.
Heat degradation: a solution of 50 µg/mL of cefiderocol was exposed to a temperature of 80°C for 2 hours. The solution was analysed directly without dilution.
Photolytic degradation: a 50 µg/mL cefiderocol solution was exposed to a UV source of 254 nm for 30 min. The solution was analysed directly without dilution.
The specificity of our method was evaluated with the analysis of NS, D5W and water for injection solutions. A 100 µg/mL solution of 5-hydroxymethylfurfural, the main degradation product of dextrose, was also analysed as it has a maximum absorbance of 284 nm. A solution of sodium tosylate at 100 µg/mL and sucrose was also analysed, as this is the main excipient found in the manufacturer’s powder.5
Physical stability
Physical stability was defined as the absence of particulate formation, haze, colour change and gas evolution. The samples were visually inspected against a white/black background with unaided eye at each analysis time. The subvisual aspect was assessed by using a UV spectrophotometer (Safas Monaco UV m²). The absorbance was measured at 350, 410 and 550 nm as recommended by the guidelines of the European consensus conference.13 Physical stability was also assessed by performing a particulate contamination test (Particle counter; PAMAS SVSS) at the beginning and end of the study. According to the criteria of the European Pharmacopoeia, preparations of a volume <100 mL comply with the particulate contamination test if the number of particles measured does not exceed 6000 particles of ≥10 µm per container and 600 particles of ≥25 µm per container.14 These criteria are used for polypropylene syringes which have a volume less than 100 mL.
Study design
Stability was monitored according to the French Society of Clinical Pharmacy (SFPC), the Group of Evaluation and Research for Protection in Areas Under Control (GERPAC) recommendations and ICH guidelines.11 12 The stability of cefiderocol was determined at four time points: after reconstitution (T0 hours) and then at T12, T24 and T48 hours. According to these recommendations, for each storage condition three preparations were produced (ie, six syringes: three diluted in NS and three diluted in D5W), stored at 20–25°C, exposed to light. Six other syringes were prepared under the same conditions (three syringes diluted with NS and three with D5W), stored at 20–25°C but protected from light. The concentrations were expressed as a percentage of the initial concentration of the preparation. At each analysis time for each preparation, the chemical stability was determined by our analytical method; all samples were analysed in triplicate. For HPLC analysis, 200 µL of the polypropylene syringe solution (62.5 mg/mL) was diluted with ultrapure water in a volumetric flask (final volume 200 mL) to obtain a theoretical concentration of 62.5 µg/mL cefiderocol. At each analysis time, a visual and subvisual inspection (UV spectrophotometry) was carried out, as well as a pH measurement with a pH meter (Bioblock Scientific model 93313). The particle count was carried out with an additional polypropylene syringe after the chemical stability period had been defined, for cost reasons and volume required to perform the examination.
Solutions were considered stable if the concentration of cefiderocol remained above 90% of the initial concentration. The maximum pH variation accepted during the study was 1 pH unit. No physical instability should be detectable either visually or subvisually (UV spectrophotometry and particle counting).
Results
HPLC assay
The retention time for cefiderocol was 5.95 min (figure 2). The UV spectrum of cefiderocol is shown in figure 2, which has an absorbance maximum at 195 and 260 nm.
Chromatogram of cefiderocol after reconstitution and UV spectrum.
The results of the calibration study highlight the homogeneity of the variance (Cochran’s test: Cexp=0.424 <Cth(5%; 3; 5)=0.684). Linearity was demonstrated for cefiderocol with an R² of >0.9999. ANOVA (non-linearity) demonstrated the good fit of a linear model for our method (F e xp=0.07 <Fth(5%; 3; 10)=3.71). All intra-day and inter-day precisions were less than 2%.
The set of forced degradations performed is shown in figure 3 and table 1; a total of 20 degradation products were detected. Only degradation products nos 2 and 13 were found whatever the degradation mode. We were able to degrade cefiderocol between 12% with acid degradation and up to 44% with photolytic degradation. For alkaline, oxidative and photolytic degradation, a cefiderocol degradation product has the same retention time as tosylate, with an increase in its surface area compared with an immediately reconstituted solution. Only degradation product no 11 on acid degradation showed co-elution (imperfect resolution, with an Rs <1.5) with the cefiderocol peak (figure 2A). The mass balance for alkaline, heat and photolytic degradation showed a significant decrease in mass compared with a solution after reconstitution. Conversely, the mass balance was higher for oxidative degradation.
Chromatograms of cefiderocol after various forced degradations.
Mass balance of cefiderocol solutions after reconstitution and after various methods of forced degradation
In terms of specificity, the peaks detected were 5-hydroxymethylfurfural, tosylate (figure 2) and sucrose, which had a retention time of 1.8, 2.9 and 0.9 min, respectively, with our method.
Chemical stability
The remaining percentage of the initial concentration of cefiderocol with our analytical method is shown in table 2. In polypropylene syringes, cefiderocol retains more than 90% of its initial concentration after 12 hours of storage in NS and D5W. After 24 hours all syringes except one (syringe diluted in NS exposed to light, remaining concentration: 89.9%) retained more than 90% of the initial concentration. After 48 hours, no solution retained more than 90% of the initial concentration regardless of the solvent or light exposure (82.9–89.4%).
Chemical stability of cefiderocol diluted in 0.9% sodium chloride (NS) or in dextrose 5% in water (D5W)
Regarding pH measurements, after 12 hours the maximum variation observed was 0.03 units and the maximum variation observed during the stability study was at 48 hours with a variation of 0.1 unit (5.22–5.32) for a syringe diluted with D5W exposed to light.
Physical stability
After reconstitution and preparation of the polypropylene syringes the solutions were clear and colourless. Throughout the study no physical changes were detected, either the formation of gas or precipitate, or the detection of a colour change.
The subvisual analysis by UV spectrophotometry is shown in table 3. This analysis shows no change in absorbance at 350 nm or at 550 nm. At 410 nm the absorbance increases over time, with a greater increase observed after 48 hours for the light-exposed syringes, but at 12 hours there were no observable differences between the syringes according to their light exposure.
Absorbance values for cefiderocol solutions during the stability study
Particle counting was performed on a syringe of cefiderocol diluted in NS and D5W exposed to light with analysis at T0 and T12 hours. This average count is related to the initial syringe volume of 48 mL. For the syringe diluted in NS, 352 particles ≥10 µm and 16 particles ≥25 µm were counted on average after reconstitution. After 12 hours, 896 particles ≥10 µm and 48 particles ≥25 µm had been counted. For the syringe diluted in D5W, 256 particles ≥10 µm and 32 particles ≥25 µm were counted on average after reconstitution. After 12 hours, 1312 particles ≥10 µm and 16 particles ≥25 µm had been counted. The particle count was in accordance with the standards set by the European Pharmacopoeia.
Discussion
Our results show that the methods developed were reliable and adequate for the assessment of the physicochemical stability of cefiderocol based on selectivity, linearity, sensitivity, precision and accuracy. To the best of our knowledge, this study is the first to have developed and validated a stability indication method for assessing the chemical stability of cefiderocol. During our study only peak no 2 was detected; peak no 11 was not detected.
The subvisual analysis is intended to verify the absence of particle formation. It was confirmed by particle counter analysis and by measuring the absorbance at 550 nm. The relative increase in absorbance at 410 nm may be due to the presence of a degradation product, but spectrum analysis of degradation product no 2 does not reveal absorption at this wavelength. This may be because our method does not detect a possible degradation product as the mass balance performed during forced degradation reveals a loss of mass for alkaline, heat and photolytic degradation. This may be due to the loss of chromophore grouping during the degradation of cefiderocol. Conversely, the mass balance was increased with oxidative degradation, since the H2O2 used for degradation absorbs (degradation product no 1) with this method.
This was the first stability study carried out on cefiderocol. We show that the solvents used (NS or D5W) had no influence on the stability. Exposure to light also had no influence on stability, although exposure to UV light for a short time (30 min) during forced degradation degraded about 50% of the cefiderocol present in solution.
We cannot compare this stability time with other studies on cefiderocol, but we can compare it with two cephalosporins which share a very similar chemical structure: ceftazidime and cefepime.15 Arsene et al were able to demonstrate a 20-hour stability at 20°C for solutions of ceftazidime diluted in NS or D5W at 40 mg/mL in polypropylene bags.16 Nahata et al demonstrated 24-hour syringe stability for ceftazidime diluted in water for injection at 100 mg/mL and stored at 22°C.17 For cefepime, Stewart et al showed 48-hour stability at 20°C for solutions of ceftazidime diluted in NS or D5W at 200 mg/mL in polypropylene syringes and stored at 20–22°C.18 Curti et al reported a stability in polypropylene syringes of 10 hours for solutions diluted in NS at 120 mg/mL and stored at 25°C.19 Although it is difficult to compare stability times with different molecules, our study found similar results (with similar concentrations, temperatures and containers) for antibiotics belonging to the same cephalosporin family. In view of the results of the chemical stability (HPLC), all points are >90% of the initial concentration except for one syringe at 24 hours (89.94±1.28%). As a precaution, we will suggest 12 hours of stability, but the degradation kinetics allow time to be added for possible preparation in a centralised unit and immediate transport to the patient.
Conclusion
In polypropylene syringes, cefiderocol 62.5 mg/mL (3 g in 48 mL) diluted in NS or D5W was stable for 12 hours at room temperature. Exposure to light had no influence on the physicochemical stability. These new data allow the use of cefiderocol in a continuous infusion for patients in ICUs.
What this paper adds
What is already known on this subject
Cefiderocol is a new siderophore time-dependent antibiotic of last resort.
The manufacturer reports a stability of 6 hours for the infusion solution.
Optimising its effectiveness by continuous infusion is therefore crucial.
What this study adds
This is the first stability study on cefiderocol.
In polypropylene syringes, cefiderocol 62.5 mg/mL is stable for 12 hours at room temperature.
Exposure to light or the choice of diluent (sodium chloride or dextrose 5% in water) has no influence on the physicochemical stability.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
Ethics statements
Patient consent for publication
Acknowledgments
The authors thank Jacques Kuhnlé for reading through the manuscript and making corrections, and Franck Blaise, Nathalie Sobalak and Hubert Zenier for their technical assistance and their help during this study.
Footnotes
EAHP Statement 3: Production and Compounding. EAHP Statement 4: Clinical Pharmacy Services.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.