Physicochemical stability of Methotrexate Accord in punctured original vials and after dilution with 0.9% sodium chloride or 5% glucose solution in non-PVC bags

Author byline as per print journal: Irene Krämer, PhD; Judith Thiesen, PhD

Study objectives: To determine the physicochemical stability of Methotrexate Accord 25 mg/mL concentrate in punctured original vials and after dilution with 0.9% sodium chloride or 5% glucose solution in 100 mL non-PVC bags containing 0.1 mg/mL and 20 mg/mL methotrexate stored at different temperature conditions for a maximum period of 56 days.
Methods: Test solutions were prepared by using Methotrexate Accord 25 mg/mL and stored at 20°C–25°C without light protection (concentrate and diluted infusion solutions) or light protected at 2°C–8°C (diluted infusion solutions). Diluted infusion solutions were prepared by addition of the methotrexate concentrate to non-PVC infusion bags pre-filled with 0.9% sodium chloride or 5% glucose solution. Samples were taken and analysed initially (Day 0) and at predetermined time points. Analysis comprised pH measurements, visual inspections, and high-performance liquid chromatography (HPLC) assays, including detection of related substances.
Results: Methotrexate Accord concentrate in punctured vials remained physicochemically stable when stored at room temperature without light protection for 56 days.
Diluted methotrexate 20 mg/mL solutions in non-PVC bags remained physicochemically stable for 56 days, independent of the type of vehicle solution and storage conditions. Methotrexate infusion solutions 0.1 mg/mL remained stable over the 2-day study period at room temperature (normal room light) and 56-day study period at 2°C–8°C (light protected), respectively.
Conclusion: Remaining solution of Methotrexate Accord 25 mg/mL in punctured vials can be used for 56 days when stored at room temperature. Diluted Methotrexate Accord infusion solutions 20 mg/mL in non-PVC bags can be prepared in advance and used over a period of 56 days, irrespective of the type of vehicle solution (0.9% sodium chloride, 5% glucose solution) and storage conditions. Physicochemical stability of diluted Methotrexate Accord 0.1 mg/mL solutions is established over the observation period of 2 days at room temperature and 56 days under refrigeration.

Submitted: 15 April 2024; Revised: 25 April 2024; Accepted: 2 May 2024; Published online first: 13 May 2024

Introduction

Methotrexate is an antifolate antineoplastic agent that inhibits the synthesis of purine and pyrimidine bases,thereby hindering DNA synthesis. It is primarily used to treat acute lymphocytic leukaemia, non-Hodgkin’s lymphoma, osteogenic carcinoma, and breast cancer. Because of its immunosuppressive activity, methotrexate is indicated for the treatment of severe psoriasis or rheumatoid arthritis. Methotrexate 25 mg/mL was reported to be physicochemically stable in the original vial after first opening for 28 days,whether stored refrigerated or at room temperature [1]. Methotrexate infusion solutions in the concentration range of 0.1 mg/mL to 20 mg/mL were proven to be stable for up to 30 days, irrespective of the vehicle solution (0.9% sodium chloride, 5% glucose solution),and storage temperature [2–4]. According to the Summary of Product Characteristics (SmPC), physicochemical stability of diluted Methotrexate Accord solutions is given for 24 hours [5]. Any unused product in the punctured original vial should be discarded [5].

Study objectives

In this study,the physicochemical stability of Methotrexate Accord 25 mg/mL concentrate should be determined in original vials after first opening and after dilution with 0.9% sodium chloride or 5% glucose solution in non-PVC bags containing 0.1 mg/mL and 20 mg/mL methotrexate,stored under different storage conditions for a maximum period of 56 days.

Methods

Methotrexate test solutions were prepared under EU Class A conditions and in accordance with the principles of Good Manufacturing Practice. A total of five different test solutions were prepared using the European Medicines Agency (EMA) licensed Methotrexate Accord 25 mg/mL (batch number P02319). Methotrexate Accord concentrate was stored at 20°C –25°C without light protection. Diluted test solutions were stored light protected in the refrigerator (2°C –8°C ) or without light protection at room temperature (20°C –25°C ). Samples were taken and analysed initially (Day 0) and at predetermined time points. For detailed information, see Table 1.

Table 1

The physical stability analysis of all test solutions was assessed by pH measurement ( using a glass electrode calibrated with standard buffer solutions) and visual inspection under standard laboratory light for any change s in colour, clarity,or the presence of particulate matter.

Chemical stability was determined via high-performance liquid chromatography (HPLC) analysis, which has been validated for linearity of analytical response and acceptable precision. The assay was proven to be stability-indicating for non-specific degradation of the parent drug [6]. Acceptance criteria were set for a methotrexate concentration of 95%–105% of the label claim for methotrexate concentrate in punctured vials and 90%–110% of the initially measured methotrexate concentration for all diluted test solutions [6]. The t ype and quantity of related substances were analysed by HPLC. Peak areas of related substances were calculated as a percentage rate of the intact methotrexate peak. Acceptance criteria for the peak areas were set as follows [6]:

Methopterin (Impurity C): </= 3.0%
Other single known impurity: </= 0.2%
Single unknown impurity: </= 0.1%
Total impurities other than Impurity C: </= 1.0%

Results

Methotrexate Accord 25 mg/mL concentrate in punctured vials,stored at room temperature without light protection,remained physicochemically stable over the observation period of 56 days. Neither colour change, turbidity,nor visible particles were observed during visual inspection. The HPLC assays revealed only slight variations of methotrexate concentrations over time,which are considered to be related to assay variability, see Table 2. Several peaks of related substances were observed in the chromatograms of samples taken initially and over the 56-day period. Peak areas of the known and unknown impurities were well below the specified limits (e.g. methopterin maximum 0.343% of the main peak, with a limit of 3.0%).

Table 2

Diluted Methotrexate Accord 20 mg/mL solutions in non-PVC bags remained physicochemically stable for 56 days, irrespective of vehicle solution (0.9% sodium chloride, 5% glucose solution) and storage condition, as shown in Table 3. The p eak areas of related substances detected in the chromatograms remained below the acceptance criteria, as described in method section.

Table 3

Diluted Methotrexate Accord 0.1 mg/mL solutions remained physicochemically stable over the 2-day observation period when stored at room temperature under ambient light conditions,and over the 56-day observation period when stored refrigerated, irrespective of vehicle solution used (0.9% sodium chloride, 5% glucose solution), as shown in Table 4. All peaks of related substances detected in the chromatograms remained below the acceptance criteria described in Methods section.

Table 4

Conclusion

Methotrexate Accord solution concentrate 25 mg/mL stored at room temperature remained physicochemically stable over the 56-day observation period after first opening. After dilution with 0.9% sodium chloride or 5% glucose infusion solution,Methotrexate Accord 0.1 mg/mL and 20 mg/mL solutions were proven to be physicochemically stable over the observation period of 56 days when stored refrigerated. Methotrexate Accord 0.1 mg/mL solutions remained physicochemically stable over the 56-day observation period when stored refrigerated and over the 2-day observation period when stored at room temperature.

Unused portions in punctured original vials can be used cost-effectively for 56 days when stored at room temperature. Diluted Methotrexate Accord infusion solutions,0.1 mg/mL up to 20 mg/mL in non-PVC bags,can be prepared in advance by pharmacy-based cytotoxic preparation units and used over a period of 56 days when stored light protected at 2°C–8°C.

Analysis was performed and documented by an accredited external laboratory. Results were carefully checked for plausibility and cautiously interpreted.

Funding sources

This study was funded by Accord Healthcare.

Competing interests: The authors Irene Krämer, Frank Erdnuess, and Judith Thiesen have no competing interest s to declare.

Provenance and peer review: Not commissioned; externally peer reviewed.

Authors

Professor Irene Krämer, PhD
Frank Erdnuess, PhD
Judith Thiesen, PhD

Department of Pharmacy, University Medical Center of the Johannes Gutenberg University Mainz, 1 Langenbeckstraße, DE-55131 Mainz, Germany

References
1. Klein M, Carstens G. Stabilität und Kompatibilität von Methotrexat-Lösungen. Onkologische Pharmazie. 2004;3:2-3.
2. Dyvik O, Grislingaas AL, Tonnesen HH, Karlsen J. Methotrexate in infusion solutions – a stability test for the hospital pharmacy. J Clin Hosp Pharm. 1986;11(5):343-8.
3. Nissen KB, Jorgensen LB, Berg DL, Andersen G. Stability study of methotrexate in 0.9% sodium chloride injection and 5% dextrose injection with limit tests for impurities. Am J Health Syst Pharm. 2017;74(9): e211-e223.
4. Benaji B, Dine T, Goudaliez F, Luyckx M, Brunet C, Mallevais ML, et al. Compatibility study of methotrexate with PVC bags after repackaging into two types of infusion admixtures. Int J Pharmaceut. 1994;105:83-7.
5. Accord Healthcare Limited. Summary of product characteristics for Methotrexate Accord 25 mg/mL concentrate for solution for infusion. Available from: https://www.accord-healthcare.com/ie/system/files/spc/ie-SPC-clean-v040%20-%20pfs.pdf
6. Accord Healthcare Limited. Data for HPLC assay and acceptance criteria on file; 23-08-13.

Author for correspondence: Judith Thiesen, PhD, Department of Pharmacy, University Medical Center of the Johannes Gutenberg University Mainz, 1 Langenbeckstraße, DE-55131 Mainz, Germany

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