Fused-core HPLC method development implemented in a short-term stability study of Triple IT solution

For the majority of children with acute lymphoblastic leukemia (ALL), treatment consists in part of a triple intrathecal (Triple IT) therapy, i.e. a combination of cytarabine (CB), methotrexate (MTX) and methylprednisolone sodium succinate (MPSS) [1]. This combination product is prepared ex-tempore. However, no in-use shelf-life under defined storage conditions has yet been established. During stability studies, a large number of samples are generated, thus creating the need for a fast, accurate and selective analytical method. In this study, a fused-core HPLC method was developed. This hybrid technology, consisting of a 0.5 µm thick porous shell fused to a 1.7 µm inert core, enables faster chromatographic separation with sufficiently high resolution. During method development, both stressed and unstressed solutions containing both single Triple IT components and the mixture thereof, were analyzed using different linear gradient times, ranging from 5 to 30 min. The mobile phase composition was fixed (A: 0.1% glacial acid in H2O; B: 0.1% glacial acid in ACN), starting with A:B (90:10, V/V) and ending with A:B (10:90, V/V). Method selectivity was evaluated based on the observed peaks in stressed CB, MTX and MPSS solutions, i.e. incubation at 40°C and 80°C. A balance between fast separation and sufficient resolution between the Triple IT components and related degradants, was found by setting the gradient time at 15 min. The Triple IT related degradation peaks were chromatographically separated from the remaining Triple IT components. Moreover, selectivity was supported by a peak purity analysis on the observed peaks. Linearity was demonstrated (R² > 0.999) for the three Triple IT components. Repeatability was evaluated by triplicate injections of 100% reference assay: relative standard deviation varied between 0.155% (MPSS), 0.464% (CB) and 1.352% (MTX) [2]. References [1] A. Ruggiero, V. Conter, M. Milani, E. Biagi, I. Lazzareschi, P. Sparano, R. Riccardi. Intrathecal chemotherapy with antineoplastic agents in children. Paediatric drugs 3(4) (2001) 237-246. [2] M. D’Hondt, E. Vangheluwe, S. Van Dorpe, J. Boonen, T. Bauters, B. Pelfrene, J. Vandenbroucke, H. Robays, B. De Spiegeleer. Stability of ex-tempore prepared Triple intrathecal solution consisting of cytarabine, methotrexate and methylprednisolone sodium succinate. American Journal of Health-System Pharmacy, submitted for publication

Short-term storage stability of Triple IT solution

For the majority of children with acute lymphoblastic leukemia (ALL), treatment consists in part of a triple intrathecal (Triple IT) therapy, i.e. a combination of cytarabine (CB), methotrexate (MT) and methylprednisolone sodium succinate (MPSS) [1]. This combination product is prepared ex-tempore. However, no in-use shelf-life under defined storage conditions has yet been established. In this study, the short-term storage stability (up to 48 hours) was evaluated and included following study design variables: i) 3 packaging materials, i.e. PhaSeal® system, dark glass vial and dark glass vial + needles, ii) 4 storage conditions, i.e. light protected at 5 °C, 25 °C, 40 °C and photometric (UV and visible light) exposure at 25 °C, iii) batch reproducibility, i.e. 3 different batches. Calculation of the degradation kinetic parameters, obtained from HALO-HPLC-UV/DAD data, was performed using the Arrhenius equation to describe the temperature dependence. Moreover, identity of the major degradants was performed using ESI-MSn detection, and their specification relevance discussed. While CB is reasonably stable, MPSS did show hydrolysis as well as isomerisation (21- to 17-succinate ester). Moreover, MT was extremely light sensitive under the tested ICH conditions. The recommended storage condition for the ex-tempore prepared Triple IT solution is in a refrigerator (5 ± 3 °C) and protected from light. Primary packaging can be PhaSeal® syringes as well as glass vials. Under these conditions, no functionality-significant degradation is expected over a 24 hour storage period [2]. References: [1] A. Ruggiero, V. Conter, M. Milani, E. Biagi, I. Lazzareschi, P. Sparano, R. Riccardi. Intrathecal chemotherapy with antineoplastic agents in children. Paediatric drugs 3(4) (2001) 237-246. [2] M. D’Hondt, E. Vangheluwe, S. Van Dorpe, J. Boonen, T. Bauters, B. Pelfrene, J. Vandenbroucke, H. Robays, B. De Spiegeleer. Stability of ex-tempore prepared Triple intrathecal solution consisting of cytarabine, methotrexate and methylprednisolone sodium succinate. American Journal of Health-System Pharmacy, submitted for publication

Stability of extemporaneously prepared cytarabine, methotrexate sodium, and methylprednisolone sodium succinate

Purpose. The short-term stability of extemporaneously prepared triple intrathecal therapy, containing cytarabine, methotrexate sodium, and methylprednisolone sodium succinate, was evaluated. Methods. Three batches of triple intrathecal solution were prepared using commercially available products and stored in three different packaging materials (plastic syringe system, brown glass vials, and brown glass vials filled with metal needles). The solutions were protected from light and stored at 5 degrees C, 25 degrees C, and 40 degrees C or exposed to ultraviolet and visible light at 25 degrees C, compliant with the International Conference on Harmonisation. Samples were taken immediately before and after 4, 8, 24, 32, and 48 hours of storage. Simultaneous high-performance liquid chromatography-ultraviolet light/diode array detector assay of cytarabine, methotrexate sodium, and methylprednisolone sodium succinate was performed using a fused-core stationary phase and an acetonitrile-based gradient. First-order kinetic degradation values were calculated, and temperature dependence was evaluated using the Arrhenius equation. Results. Cytarabine was stable under all storage conditions. Methotrexate sodium displayed significant degradation after light exposure but remained stable under the other storage conditions. Methylprednisolone sodium succinate was found to be the most labile component in the triple intrathecal solution. Temperature-dependent degradation was observed, resulting in 46% degradation after 48 hours at 40 degrees C. Two degradants were formed: methylprednisolone and methylprednisolone hydrogen succinate. Packaging material and batch-to-batch variability did not significantly influence the stability of the triple intrathecal solution. Conclusion. Triple intrathecal solution of cytarabine, methotrexate sodium, and methylprednisolone sodium succinate was stable for up to 12 hours when stored at 5 degrees C and protected from light