TY - JOUR
AU - , De Spiegeleer, Bart
AB - Abstract 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 °C, 25 °C, and 40 °C or exposed to ultraviolet and visible light at 25 °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 °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 °C and protected from light. Antineoplastic agents, Chromatography, liquid, Compounding, Containers, Contamination, Cytarabine, Glass, Injections, Methotrexate sodium, Methylprednisolone, Methylprednisolone hydrogen succinate, Methylprednisolone sodium succinate, Photodecomposition, Plastics, Stability, Steroids, cortico, Storage, Temperature Prevention of meningeal leukemia is a complex problem. As the blood-brain barrier limits the availability of intravenously administered drugs, intrathecal administration of chemotherapy has become a keystone of treatment protocols for acute lymphoblastic leukemia or lymphoma.1–3 Intrathecal chemotherapy is mainly used to treat leukemia and lymphoma in children and selected brain tumors. In these diseases, routine therapeutic use of intrathecal chemotherapy is limited to methotrexate and cytarabine, which must be accompanied by a corticosteroid.4 Intrathecal administration of methotrexate, a synthetic inhibitor of dihydrofolate reductase, and cytarabine, a pyrimidine anti-tumor agent, may cause neurotoxic effects such as vomiting, fever, paraplegia, and meningoencephalopathy. Simultaneous administration of a corticosteroid (e.g., hydrocortisone, methylprednisolone) can significantly reduce these complications.5 However, as hydrocortisone preparations without benzyl alcohol are not available worldwide and because benzyl alcohol is contraindicated for intrathecal administration, methylprednisolone sodium succinate has become standard in hospital intrathecal mixtures. However, no stability information for this triple intrathecal mixture is currently available.6 The purpose of this study was to evaluate the stability of methotrexate, cytarabine, and methylprednisolone sodium succinate in solution, according to International Conference on Harmonisation (ICH) guidelines, up to 48 hours after extemporaneous preparation.7 The potential influence of different packaging materials and batch-to-batch variability was also investigated. Methods Preparation of triple intrathecal solutions Three batches of triple intrathecal solutions were prepared by transferring 11.94 mL of 2-mg/ mL methylprednisolone sodium succinate in 0.9% sodium chloride injectiona into a sterile brown vial.b Subsequently, 38.22 mL of 2.5-mg/ mL methotrexate (as the sodium salt)c and 11.94 mL of 20-mg/mL cytarabine solutiond were added to obtain the triple intrathecal solution, thus containing 0.051% m/v, 0.154% m/v, and 0.385% m/v of aforementioned drug substances, respectively. Finally, the triple intrathecal solution was added to three different packaging materials that are used during intrathecal administration: plastic syringes,e brown glass vials,f and brown glass vials in which metal needlesg are placed. This last condition mimics the worst-case situation of the solution contacting the needle. Preparation of reference solutions Reference solution 1, used for high-performance liquid chromatographic (HPLC) assay of methylprednisolone sodium succinate, was prepared by dissolving 2.20 mg methylprednisolone sodium succinate,h 7.65 mg methotrexatei (as the sodium salt), and 19.35 mg cytarabine,j using 5 mL of 130 mM phosphate buffer (pH 7),k,l in a 10-mL volumetric flask. The solution was diluted to volume by a solvent mixture consisting of 90% purified waterm and 10% acetonitrile.n Reference solution 2, used for HPLC assay of cytarabine and methotrexate, was prepared by diluting 1 mL of reference solution 1 to 20 mL, using the water–acetonitrile solvent mixture. Finally, 2.20-mg/mL methylprednisolone sodium succinateh and methylprednisoloneo single active pharmaceutical ingredient (API) reference solutions, used for liquid chromotography–mass spectrometry (LC-MS) identification of methylprednisolone sodium succinate-related degradants, were prepared using the water– acetonitrile solvent mixture. Stability study Temperature stability and potential packaging influence were evaluated by storage of the three triple intrathecal batches, added to the aforementioned three packaging materials, at 5 °C (50% relative humidity), 25 °C (60% relative humidity), and 40 °C (75% relative humidity), all protected from light and compliant with ICH guidelines. Needle-filled brown glass vials were stored at 40 °C only. Samples were taken immediately after preparation and at 4, 8, 24, 32, and 48 hours. Exact time intervals (within 20% of these preset time points) were used to calculate the chemical degradation rates of methylprednisolone sodium succinate, methotrexate, and cytarabine. Test solution 1, used for the methylprednisolone assay, was prepared by diluting 1 mL of triple intrathecal solution to 2 mL using the water–acetonitrile solvent mixture. Test solution 2, used for the cytarabine and methotrexate assays, was prepared by diluting 1 mL of test solution 1 to 20 mL using the water–acetonitrile solvent mixture. In total, 114 samples were evaluated (three batches × three tinitial measurements) + (three batches × seven storage conditions × five time points). The photochemical stability of batches 1 and 3 was evaluated by incubation under ultraviolet light (six fluorescent lamps F18 W/BLB-T8p for two days and visible light (six cool white fluorescent lamps TDL 18 W/33q for eight days. Photochemical stability was evaluated by packaging triple intrathecal solution in quartz cuvettesr; control solutions were protected from light by wrapping the cuvettes with aluminium foil. Temperature and relative humidity were set at 25 °C and 60%, respectively. The aforementioned storage conditions complied with ICH requirements and actinometric qualification.7 In total, 8 samples were evaluated (2 [ultraviolet light and visible light] × 2 [light exposed and light protected] × two batches). HPLC analysis Assay of the triple intrathecal solution components and possible degradants was performed using an HPLC systems equipped with a separation modulet and photodiode-array detector.u,v Chromatographic separation was achieved using a fused-core, reverse-phase column,w preceded by a guard columnx and heated to 25–35 °C. Mobile phases consisted of 0.1% glacial acetic acidy in waterm and 0.1% glacial acetic acid in acetonitrile. The flow rate was set at 1.0 mL/ min, and the linear gradient was as follows: 0.1% glacial acetic acid in water:0.1% glacial acetic acid in acetonitrile (90:10, v/v) at 0, 20, and 30 minutes and 0.1% glacial acetic acid in water:0.1% glacial acetic acid in acetonitrile (10:90, v/v) at 15 and 18 minutes. The temperature of the sample compartment was set at 10–20 °C. The ultraviolet-light detector was set at 240 nm for the methyl-prednisolone assay and at 280 nm for the cytarabine and methotrexate assays. The injection volume was 10 μL. The LC-MS apparatus, used for identification of the major degradants, consisted of an HPLC interface,z a solvent degasser,aa a pump,bb an autosampler,cc and an ion-trap mass spectrometerdd in positive-ion mode equipped with a dual-wavelength absorbance ultraviolet-light detector.ee Xcalibur 2.0 software (Thermo, San Jose, CA) was used during data acquisition and processing. Electrospray ionization was conducted using a needle voltage of 4.5 kV. Nitrogen was used as the sheath and auxiliary gas, with the heated capillary set at 250 °C. Positive-mode mass spectra were obtained in a mass-to-charge ratio of 50–1000 m/z. HPLC validation An HPLC method was developed using a fused-core silica stationary phase.8 This hybrid technology, consisting of a 0.5-μm thick porous shell fused to a 1.7-μm inert core,w enables faster chromatographic separation with similar resolution to classic HPLC columns, which use fully porous particles. During method development, both stressed and unstressed solutions containing single triple intrathecal components and the mixture of all three components were analyzed using different linear gradient times, ranging from 5 to 30 minutes. The mobile phase composition was fixed, starting with 0.1% glacial acetic acid in water:0.1% glacial acetic acid in acetonitrile (90:10, v/v) and ending with 0.1% glacial acetic acid in water:0.1% glacial acetic acid in acetonitrile (10:90, v/v). A balance between fast separation and sufficient resolution between the triple intrathecal components and related degradants was found by setting the gradient time at 15 min. Method selectivity was evaluated based on the observed peaks in stressed cytarabine, methotrexate, and methylprednisolone solutions (i.e., incubated for 29 hours at 40 °C and 80 °C). The degradation peaks were chromatographically separated from the remaining triple intrathecal components. Moreover, selectivity was supported by a peak purity analysis of the observed peaks. Linearity (r2 > 0.999) was demonstrated for the triple intrathecal components. Repeatability was evaluated by triplicate injections of 100% reference assay. Relative standard deviations were 0.16% for methylprednisolone sodium succinate, 0.46% for cytarabine, and 1.35% for methotrexate sodium. Data analysis The stability of the triple intrathecal components was assessed by comparing the percentage of initial concentration remaining at each time interval. To support these results, peak area balance was calculated and compared with the peak area at t0. When statistically significant degradation (p < 0.05) was observed, the degradation constant k was calculated from linear regression data, assuming first-order degradation kinetics. In this study zero, first, and second order kinetic models were compared and it was found that the first order degradation model best fit the experimental data. Frequency factor and activation energy of the degradation reaction were derived from the Arrhenius9 equation. Multiple linear regression analyses, expressing k as a function of batch and packaging material with incubation at the same storage temperature, were used to elucidate the significance of batch or packaging material. The a priori level of significance was 0.05. LC-MS degradant identification was conducted by comparing the retention time and the observed molecular (MS1) and fragment (MS2) ions of the reference solutions and selected stability samples. Results Temperature stability No degradation of cytarabine or methotrexate was observed during the two-day incubation period at the different storage conditions. However, significant degradation of methylprednisolone was seen throughout all storage conditions (Table 1), combined with a consistent degradation profile (i.e., increase of two structurally related degradants). Multiple linear regression, expressing the methylprednisolone degradation rate k as a function of batch and packaging material at the same storage temperature, demonstrated no significant influence of packaging material and batch number. Based on aforementioned findings, an Arrhenius regression model was constructed using a pooled data set (three batches, three packaging materials, three incubation temperatures) (i.e., 21 k values). The activation energy and frequency factor of the methylprednisolone degradation reaction were 45.5 kJ/mol (95% confidence interval (CI), 41.6–49.4 kJ/mol) and 524,470 hr−1 (95% CI, 106,937–2,571,500 hr−1), respectively. From this, the methylprednisolone sodium succinate degradation was predicted for other relevant storage conditions (Table 2). Table 2 Calculated Methylprednisolone Sodium Succinate Degradation at Storage Temperatures Storage Temperature and Storage Time % Degradation 5 °C  1 hr 0.15  3 hr 0.45  6 hr 0.89  12 hr 1.78  24 hr 3.52  48 hr 6.92 25 °C  15 min 0.14  30 min 0.28  1 hr 0.56  1.5 hr 0.84  2 hr 1.11 37 °C  5 min 0.09  10 min 0.19  15 min 0.28  30 min 0.57 Storage Temperature and Storage Time % Degradation 5 °C  1 hr 0.15  3 hr 0.45  6 hr 0.89  12 hr 1.78  24 hr 3.52  48 hr 6.92 25 °C  15 min 0.14  30 min 0.28  1 hr 0.56  1.5 hr 0.84  2 hr 1.11 37 °C  5 min 0.09  10 min 0.19  15 min 0.28  30 min 0.57 Open in new tab Table 2 Calculated Methylprednisolone Sodium Succinate Degradation at Storage Temperatures Storage Temperature and Storage Time % Degradation 5 °C  1 hr 0.15  3 hr 0.45  6 hr 0.89  12 hr 1.78  24 hr 3.52  48 hr 6.92 25 °C  15 min 0.14  30 min 0.28  1 hr 0.56  1.5 hr 0.84  2 hr 1.11 37 °C  5 min 0.09  10 min 0.19  15 min 0.28  30 min 0.57 Storage Temperature and Storage Time % Degradation 5 °C  1 hr 0.15  3 hr 0.45  6 hr 0.89  12 hr 1.78  24 hr 3.52  48 hr 6.92 25 °C  15 min 0.14  30 min 0.28  1 hr 0.56  1.5 hr 0.84  2 hr 1.11 37 °C  5 min 0.09  10 min 0.19  15 min 0.28  30 min 0.57 Open in new tab Table 1 Stability of Methylprednisolone Sodium Succinate in Intrathecal Solution Containing Cytarabine, Methotrexate Sodium, and Methylprednisolone Sodium Succinate Storage Temperature and Container Mean ± S.D. Initial Drug Conc.a Mean ± S.D. % Initial Conc. Remaininga 4 hr 8 hr 24 hr 32 hr 48 hr 5 °C  Plastic syringe 105.03 ± 8.03 100.72 ± 0.99 100.38 ± 1.44 96.82 ± 1.04 95.34 ± 1.75 93.26 ± 1.85  Glass vial 105.95 ± 9.40 99.86 ± 1.48 99.18 ± 2.54 97.48 ± 1.95 95.10 ± 3.24 94.81 ± 1.81 25 °C  Plastic syringe 105.03 ± 8.03 95.91 ± 2.69 95.27 ± 3.07 86.26 ± 1.11 82.31 ± 2.47 76.81 ± 1.41  Glass vial 105.95 ± 9.40 97.27 ± 1.48 95.46 ± 3.17 90.21 ± 7.07 81.11 ± 3.07 77.55 ± 1.72 40 °C  Plastic syringe 105.03 ± 8.03 87.30 ± 3.28 81.16 ± 2.53 62.67 ± 5.68 59.87 ± 2.16 54.08 ± 1.87  Glass vial 105.95 ± 9.40 90.14 ± 0.30 81.48 ± 3.68 64.49 ± 4.01 58.65 ± 3.77 51.89 ± 0.81  Glass vial plus needles 105.75 ± 8.73 89.75 ± 1.61 81.49 ± 5.51 68.14 ± 3.93 60.76 ± 5.78 55.17 ± 3.38 Storage Temperature and Container Mean ± S.D. Initial Drug Conc.a Mean ± S.D. % Initial Conc. Remaininga 4 hr 8 hr 24 hr 32 hr 48 hr 5 °C  Plastic syringe 105.03 ± 8.03 100.72 ± 0.99 100.38 ± 1.44 96.82 ± 1.04 95.34 ± 1.75 93.26 ± 1.85  Glass vial 105.95 ± 9.40 99.86 ± 1.48 99.18 ± 2.54 97.48 ± 1.95 95.10 ± 3.24 94.81 ± 1.81 25 °C  Plastic syringe 105.03 ± 8.03 95.91 ± 2.69 95.27 ± 3.07 86.26 ± 1.11 82.31 ± 2.47 76.81 ± 1.41  Glass vial 105.95 ± 9.40 97.27 ± 1.48 95.46 ± 3.17 90.21 ± 7.07 81.11 ± 3.07 77.55 ± 1.72 40 °C  Plastic syringe 105.03 ± 8.03 87.30 ± 3.28 81.16 ± 2.53 62.67 ± 5.68 59.87 ± 2.16 54.08 ± 1.87  Glass vial 105.95 ± 9.40 90.14 ± 0.30 81.48 ± 3.68 64.49 ± 4.01 58.65 ± 3.77 51.89 ± 0.81  Glass vial plus needles 105.75 ± 8.73 89.75 ± 1.61 81.49 ± 5.51 68.14 ± 3.93 60.76 ± 5.78 55.17 ± 3.38 a Relative to label claim (%). b n = 3. Open in new tab Table 1 Stability of Methylprednisolone Sodium Succinate in Intrathecal Solution Containing Cytarabine, Methotrexate Sodium, and Methylprednisolone Sodium Succinate Storage Temperature and Container Mean ± S.D. Initial Drug Conc.a Mean ± S.D. % Initial Conc. Remaininga 4 hr 8 hr 24 hr 32 hr 48 hr 5 °C  Plastic syringe 105.03 ± 8.03 100.72 ± 0.99 100.38 ± 1.44 96.82 ± 1.04 95.34 ± 1.75 93.26 ± 1.85  Glass vial 105.95 ± 9.40 99.86 ± 1.48 99.18 ± 2.54 97.48 ± 1.95 95.10 ± 3.24 94.81 ± 1.81 25 °C  Plastic syringe 105.03 ± 8.03 95.91 ± 2.69 95.27 ± 3.07 86.26 ± 1.11 82.31 ± 2.47 76.81 ± 1.41  Glass vial 105.95 ± 9.40 97.27 ± 1.48 95.46 ± 3.17 90.21 ± 7.07 81.11 ± 3.07 77.55 ± 1.72 40 °C  Plastic syringe 105.03 ± 8.03 87.30 ± 3.28 81.16 ± 2.53 62.67 ± 5.68 59.87 ± 2.16 54.08 ± 1.87  Glass vial 105.95 ± 9.40 90.14 ± 0.30 81.48 ± 3.68 64.49 ± 4.01 58.65 ± 3.77 51.89 ± 0.81  Glass vial plus needles 105.75 ± 8.73 89.75 ± 1.61 81.49 ± 5.51 68.14 ± 3.93 60.76 ± 5.78 55.17 ± 3.38 Storage Temperature and Container Mean ± S.D. Initial Drug Conc.a Mean ± S.D. % Initial Conc. Remaininga 4 hr 8 hr 24 hr 32 hr 48 hr 5 °C  Plastic syringe 105.03 ± 8.03 100.72 ± 0.99 100.38 ± 1.44 96.82 ± 1.04 95.34 ± 1.75 93.26 ± 1.85  Glass vial 105.95 ± 9.40 99.86 ± 1.48 99.18 ± 2.54 97.48 ± 1.95 95.10 ± 3.24 94.81 ± 1.81 25 °C  Plastic syringe 105.03 ± 8.03 95.91 ± 2.69 95.27 ± 3.07 86.26 ± 1.11 82.31 ± 2.47 76.81 ± 1.41  Glass vial 105.95 ± 9.40 97.27 ± 1.48 95.46 ± 3.17 90.21 ± 7.07 81.11 ± 3.07 77.55 ± 1.72 40 °C  Plastic syringe 105.03 ± 8.03 87.30 ± 3.28 81.16 ± 2.53 62.67 ± 5.68 59.87 ± 2.16 54.08 ± 1.87  Glass vial 105.95 ± 9.40 90.14 ± 0.30 81.48 ± 3.68 64.49 ± 4.01 58.65 ± 3.77 51.89 ± 0.81  Glass vial plus needles 105.75 ± 8.73 89.75 ± 1.61 81.49 ± 5.51 68.14 ± 3.93 60.76 ± 5.78 55.17 ± 3.38 a Relative to label claim (%). b n = 3. Open in new tab The two related degradants were identified by means of electrospray ionization–tandem mass spectrometric detection as methylprednisolone (relative retention time [RRT], 0.88; specified impurity A) and as a methylprednisolone hydrogen succinate isomer, most likely methylprednisolone (17)-hydrogen succinate (RRT, 0.90; specified impurity B).10–14 The MS1 and MS2 spectra used to identify methylprednisolone are depicted in Figure 1. Both impurities were previously observed in a pilot methylprednisolone sodium succinate forced-degradation experiment. The two degradants were present in t Figure 1 Open in new tabDownload slide Mass spectrometry (MS)1 (top) and MS2 (bottom) spectra of methylprednisolone. Figure 1 Open in new tabDownload slide Mass spectrometry (MS)1 (top) and MS2 (bottom) spectra of methylprednisolone.0 samples: 1.92% (95% CI, 1.77–2.07%) and 1.24% (95% CI, 1.17–1.31%), respectively, based on respective peak areas relative to the methylprednisolone sodium succinate peak area and assuming a relative response factor of 1. Methylprednisolone peak area balances for all batches and time points did not significantly differ from the initial peak area balance at t0. Figure 2 depicts an overlay of typical fused-core HPLC–ultraviolet-light chromatograms obtained with test solutions 1 and 2 of triple intrathecal solution batch 1, packaged in glass vials at t Figure 2 Open in new tabDownload slide Overlay of typical fused-core high-performance liquid chromatography–ultraviolet-light chromatograms obtained from test solution 2 (assay of cytarabine and methotrexate sodium, 280 nm, lower [black]) and test solution 1 (assay of methylprednisolone sodium succinate, 240 nm, upper [red]) of triple intrathecal solution batch 1, packaged in glass vials at t0. Figure 2 Open in new tabDownload slide Overlay of typical fused-core high-performance liquid chromatography–ultraviolet-light chromatograms obtained from test solution 2 (assay of cytarabine and methotrexate sodium, 280 nm, lower [black]) and test solution 1 (assay of methylprednisolone sodium succinate, 240 nm, upper [red]) of triple intrathecal solution batch 1, packaged in glass vials at t0.0. The formation of the two methylprednisolone sodium succinate-related impurities is shown in Figure 3. No evidence of physical instability (e.g., precipitation or opalescence) was visually observed. Figure 3 Open in new tabDownload slide Overlay of typical fused-core high-performance liquid chromatography–ultraviolet-light chromatograms obtained from triple intrathecal solution batch 1 stored at 25 °C under visible light (test solution 2, assay of cytarabine and methotrexate sodium, 280 nm, lower [black]) and from triple intrathecal solution batch 1 stored at 40 °C during 48 hours (test solution 1, assay of methylprednisolone sodium succinate, 240 nm, upper [red]). RRT = relative retention time. Figure 3 Open in new tabDownload slide Overlay of typical fused-core high-performance liquid chromatography–ultraviolet-light chromatograms obtained from triple intrathecal solution batch 1 stored at 25 °C under visible light (test solution 2, assay of cytarabine and methotrexate sodium, 280 nm, lower [black]) and from triple intrathecal solution batch 1 stored at 40 °C during 48 hours (test solution 1, assay of methylprednisolone sodium succinate, 240 nm, upper [red]). RRT = relative retention time. Photochemical stability The calculated mean results for cytarabine, methotrexate, and methylprednisolone assays, expressed relative to their respective t0 assay values, are presented in Table 3. Both cytarabine and methotrexate assays in light-protected samples (i.e., incubated for two days [ultraviolet light] and eight days [visible light] at 25 °C, 60% relative humidity) were found to be similar to their respective t Table 3 Photochemical Stability of Components of Triple Intrathecal Solution Impurities Light Condition and Solution Component % Componenta Protected Stressed Ultraviolet light, exposure for 48 hrb  Cytarabine 97.59 98.85  Methotrexate sodium 98.14 65.68  Methylprednisolone (21)-sodium succinate 80.13 81.35  Methylprednisolone sodium succinate 13.58 11.72  Methylprednisolone (17)-hydrogen succinatec 9.25 8.47 Visible light, exposure for 192 hrb  Cytarabine 99.45 95.61  Methotrexate sodium 99.84 0.00d  Methylprednisolone (21)-sodium succinate 64.42 70.60  Methylprednisolone sodium succinatec 30.94 15.72   Methylprednisolone (17)-hydrogen succinatec 12.94 11.25 Light Condition and Solution Component % Componenta Protected Stressed Ultraviolet light, exposure for 48 hrb  Cytarabine 97.59 98.85  Methotrexate sodium 98.14 65.68  Methylprednisolone (21)-sodium succinate 80.13 81.35  Methylprednisolone sodium succinate 13.58 11.72  Methylprednisolone (17)-hydrogen succinatec 9.25 8.47 Visible light, exposure for 192 hrb  Cytarabine 99.45 95.61  Methotrexate sodium 99.84 0.00d  Methylprednisolone (21)-sodium succinate 64.42 70.60  Methylprednisolone sodium succinatec 30.94 15.72   Methylprednisolone (17)-hydrogen succinatec 12.94 11.25 a Mean of batch 1 (n = 2) and batch 3 (n = 2) assay results. b Conducted in accordance with the International Conference on Harmonisation Q1B guideline.7 c Calculated relative to methylprednisolone sodium succinate t0 assay. d Not detectable. Table 3 Photochemical Stability of Components of Triple Intrathecal Solution Impurities Light Condition and Solution Component % Componenta Protected Stressed Ultraviolet light, exposure for 48 hrb  Cytarabine 97.59 98.85  Methotrexate sodium 98.14 65.68  Methylprednisolone (21)-sodium succinate 80.13 81.35  Methylprednisolone sodium succinate 13.58 11.72  Methylprednisolone (17)-hydrogen succinatec 9.25 8.47 Visible light, exposure for 192 hrb  Cytarabine 99.45 95.61  Methotrexate sodium 99.84 0.00d  Methylprednisolone (21)-sodium succinate 64.42 70.60  Methylprednisolone sodium succinatec 30.94 15.72   Methylprednisolone (17)-hydrogen succinatec 12.94 11.25 Light Condition and Solution Component % Componenta Protected Stressed Ultraviolet light, exposure for 48 hrb  Cytarabine 97.59 98.85  Methotrexate sodium 98.14 65.68  Methylprednisolone (21)-sodium succinate 80.13 81.35  Methylprednisolone sodium succinate 13.58 11.72  Methylprednisolone (17)-hydrogen succinatec 9.25 8.47 Visible light, exposure for 192 hrb  Cytarabine 99.45 95.61  Methotrexate sodium 99.84 0.00d  Methylprednisolone (21)-sodium succinate 64.42 70.60  Methylprednisolone sodium succinatec 30.94 15.72   Methylprednisolone (17)-hydrogen succinatec 12.94 11.25 a Mean of batch 1 (n = 2) and batch 3 (n = 2) assay results. b Conducted in accordance with the International Conference on Harmonisation Q1B guideline.7 c Calculated relative to methylprednisolone sodium succinate t0 assay. d Not detectable.0 values. This confirms the temperature stability of these two triple intrathecal components. Cytarabine is stable under the applied ultraviolet-light conditions. However, a small amount of degradation was seen under visible light (i.e., 3.84% relative to light-protected samples; the difference between 99.45% light-protected and 95.61% light-stressed samples). This is combined with the observation of a related degradation peak at an RRT of 1.12, which was also seen in a previous forced-degradation experiment of cytarabine. The cytarabine peak area balance of the photochemical stressed samples was confirmed. Methotrexate was highly unstable under ultraviolet light (32.46% degradation). However, no significant degradation peaks were found. Under visible light, complete degradation was observed, with the emergence of two degradation peaks at RRTs of 0.85 and 1.05. Methotrexate peak area balancing proved to be unsuccessful. The reason for this may be that (1) insoluble degradants formed during light stress and remained attached to the cuvette glass wall, (2) degradants (e.g,. polymerization products) remained on the fused-core HPLC column, or (3) degradants had a significantly lower relative response factor compared with methotrexate. A yellowish precipitate, attached to the photometric exposed cuvette walls, was observed during sample preparation. Figure 3 depicts a typical fused-core HPLC–ultraviolet-light chromatogram of test solution 1 of a visible-light-stressed sample. The methylprednisolone assay results of the light-protected samples were lower than the t0 values. This was expected, given the previously observed temperature stability results. The exposure to ultraviolet and visible light did not result in additional degradation compared with the methylprednisolone assay obtained from light-protected samples. Thus, methylprednisolone is considered to be light stable. However, the peak area balance of methylprednisolone sodium succinate and the two related degradants that formed after visible light exposure was 90.83%. Furthermore, an approximate 15% decrease of methylprednisolone assay after visible light exposure was seen compared with the unstressed solution. This suggests that methylprednisolone, formed under the influence of temperature, does not remain stable in the light-stressed triple intrathecal solution. This peak area imbalance was not observed in the ultraviolet-light stressed and reference samples, where methylprednisolone assay values and peak area balance were comparable. The assay of the isomeric degradant methylprednisolone (17)-hydrogen succinate was comparable in the reference and stressed solutions after exposure to both ultraviolet and visible light. Discussion In the past, several studies have evaluated the physical and chemical stability of mixtures containing cytarabine, methotrexate, and hydro-cortisone sodium succinate, which is a different corticosteroid than the currently investigated methylprednisolone sodium succinate. Cradock et al.15 examined the hydrocortisone mixture in Elliott’s B solution, an artificial cerebrospinal fluid, at room temperature using ultraviolet-light spectroscopy. Methotrexate sodium and cytarabine were found to be stable for seven days at room temperature, but significant hydrocortisone sodium succinate degradation (7–12% loss within 24 hours) was seen. Cheung et al.,16 Zhang et al.,17 and Trissel et al.18 evaluated the cytarabine, methotrexate sodium, and hydrocortisone sodium succinate stability in Elliott’s B solution, using traditional HPLC methods. Zhang employed a separate HPLC analysis for each of the triple intrathecal components, whereas Cheung et al. and Trissel et al. used a single HPLC method, enabling simultaneous assays. Aforementioned studies demonstrated cytarabine and methotrexate stability for up to 48 hours stored at room temperature. Hydrocortisone sodium succinate degradation, ranging from 6% to 14%, was reported at room temperature. Cheung et al. identified two hydrocortisone sodium succinate degradants as the hydrolysis product (hydrocortisone) and the isomerization product (hydrocortisone [17]-hydrogen succinate). Moreover, Zhang et al. reported no stability differences due to different packaging materials. In the current study, using ICH-compliant stability conditions and a validated fused-core HPLC method for simultaneous assay, similar degradation (9.8–13.7%) of methylprednisolone sodium succinate was seen after 24 hours at 25 °C, combined with an increase in methylprednisolone (21)-sodium succinate and methylprednisolone (17)-hydrogen succinate. Furthermore, different packaging materials and batch variability did not influence the methylprednisolone sodium succinate degradation. From the acquired data (i.e., activation energy and frequency factor of methylprednisolone sodium succinate degradation), relevant in-use storage conditions were calculated (Table 2). When taking a conservative approach, storing triple intrathecal solution for a 12-hour period at 5 °C, immediately after preparation, would result in a calculated methylprednisolone degradation of 1.8%. This finding is consistent with the storage conditions given in the information leaflet of methylprednisolone sodium succinate.h,19 A 30-minute heating cycle to 37 °C immediately before intrathecal administration will cause 0.3% of additional degradation. Finally, during a 1-hour triple intrathecal administration period at room temperature (25 °C), 0.5% of methylprednisolone sodium succinate degradation will occur. Thus, manufacturing should be done in a temperature-controlled room, and solvents should be cooled before use. Although no stability data of methotrexate in this mixture are available, some information does exist concerning methotrexate as a lone component.20–23 The material safety data sheet of methotrexate lists light exposure as a cause of chemical degradation. Moreover, Chatterji and Gallelli24 observed a zero-order photolytic degradation mechanism in aqueous solution catalyzed by bicarbonate ion after incubation under fluorescent light. Furthermore, three methotrexate-related photodegradants were identified: 2,4-diamino-6-pteridinecarbaldehyde, 2,4-diamino-6-pteridinecarboxylic acid, and p-aminobenzoylglutamic acid. To confirm the previous findings, an additional photochemical stability experiment was conducted by the investigators using a reference solution containing only methotrexate. The results of this additional study are presented below. The same six time points used in the temperature stability study were chosen in order to obtain a kinetic model. The half-life of methotrexate was 6.47 and 9.04 hours under ICH-standardized visible and ultraviolet light, respectively. From this, it can be seen that methotrexate is degraded at a higher rate in the single-compound stability study when compared with the triple intrathecal stability study (i.e., t½ = 43.41 hours) under ultra-violet light. This can be explained by the absence of other ultraviolet-/ visible-light-absorbing molecules in the single-compound solution (e.g., cytarabine, methylprednisolone sodium succinate), which are present in the triple intrathecal solution, thus subjecting methotrexate to different photochemical stress conditions. Due to the 100% methotrexate degradation observed after visible light stability testing, no half-life value can be calculated. Due to the presence of salts in the pharmaceutical product, the first four minutes of the LC-MS analysis were directly diverted to waste. Hence, the methotrexate photodegradants were not characterized by LC-MS. Highest priority was given to the identification of methylprednisolone sodium succinate degradants, as their formation can be controlled only to a certain extent whereas methotrexate photodegradation is not significant when protected from light. The European Pharmacopoeia monograph of methylprednisolone hydrogen succinate describes the limits for the two degradants found in the stability samples as 0.5% individually allowed in the API.10 Hence, the acceptance limits are higher in the finished pharmaceutical product to incorporate the additional manufacturing and analytical variability (e.g., 1% individually). Assuming formation of 1% methylprednisolone in the triple intrathecal solution, no precipitation would occur. Precipitation would only occur if 31.20% methylprednisolone formed in solution. Moreover, the United States Pharmacopeia allows up to 6.6% of free methylprednisolone in methylprednisolone sodium succinate powder for injection.25 The United States Pharmacopeia monograph of methotrexate sets the individual acceptance limits for impurities at 0.5%,26 while the European Pharmacopoeia monograph describes 12 possible methotrexate-related impurities, of which 6 are specified (impurities B, C, E, F, H, and I).27 It is assumed that no methotrexate racemization occurred (no formation of impurity F) in this study. All of these specified impurities are individually limited to 0.2–0.5%. The unspecified impurity limit is set at 0.05%. The sum of all impurities, excluding specified impurities B, E (both limited to 0.3%), and C (limited to 0.5%), must be below 0.5%. From these pharmacopoeial acceptance criteria for both APIs degrading under the study conditions, it is clear that some degradation is allowed for methylprednisolone sodium succinate but not for methotrexate, thereby emphasizing the importance of light protection. Conclusion Triple intrathecal solution of cytarabine, methotrexate sodium, and methylprednisolone sodium succinate was stable for up to 12 hours when stored at 5 °C and protected from light. Footnotes The authors have declared no potential conflicts of interest. Funded by grant 101529 from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). a Solu-Medrol powder for injection, Pfizer, Puurs, Belgium, lots X06014 (batches 1 and 2) and X02490 (batch 3). b Type I WI 20, Gaasch Packaging, Mollem, Belgium, lot 70109071. c Emtrexate, Pharmachemie, Haarlem, Netherlands, lot 10A25LB. d Cytarabine, Pfizer, lots EK74F (batches 1 and 2) and ET6970 (batch 3). e PhaSeal injector Luer-Lok, Carmel Pharma, Göteborg, Sweden, lot 10020710. f 30-mL, all round brown RD 18, Gaasch Packaging, lot 70109079. g BD 18G needles, BD, Erembodegem, Belgium, lots 1009 04 (batch 1), 1009 (batch 2), and 1010 07 (batch 3). h Methylprednisolone sodium succinate, Sigma-Aldrich, Bornem, Belgium, lot 118K1241. i Methotrexate, Sigma-Aldrich, lot 079K1241. j Cytarabine, Fluka, Bornem, Belgium, lot 1352590. k Potassium phosphate dibasic, Merck, Overijse, Belgium, lot K28624480. l Potassium phosphate monobasic, Fluka, lot 1253517. m Arium 611 purification system, Sartorius, Gottingen, Germany. n Acetonitrile, Fischer Scientific, Erembodegem, Belgium, lots 1001336 and 1094240. o Methylprednisolone, ABC Chemicals, Wouters-Brakel, Belgium, lot 07K19U0055. p Philips, Eindhoven, Netherlands. q Sylvania, Yokohama, Japan. r Hellma, Knuibeke, Belgium. s Empower 2, Waters, Milford, MA. t Waters Alliance 2695, Waters. u Waters 2998 Photodiode Array Detector, Waters. v Waters 2996 Photodiode Array Detector, Waters. w HALO C18 column, 2.7-μm particle size, 4.6 × 150 mm, Achrom, Machelen, Belgium, lots AH102217 and AH102236. x HALO C18 column, 2.7-μm particle size, 4.6 × 5 mm, Achrom, lot AH102208. y Glacial acetic acid, Sigma-Aldrich, lot 227996PK. z Thermo Spectra System SN4000, Thermo, San Jose, CA. aa Spectra System SCM 1000, Thermo. bb Spectra System P1000XR, Thermo. cc Spectra System AS3000, Thermo. dd Finnigan LCQ Classic, Thermo. ee Waters 2487, Waters. 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WorldCat COPAC Copyright © 2012 by the American Society of Health-System Pharmacists, Inc. All rights reserved.
TI - Stability of extemporaneously prepared cytarabine, methotrexate sodium, and methylprednisolone sodium succinate
JF - American Journal of Health-System Pharmacy
DO - 10.2146/ajhp110208
DA - 2012-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/stability-of-extemporaneously-prepared-cytarabine-methotrexate-sodium-c2HiFFr8Un
SP - 232
VL - 69
IS - 3
DP - DeepDyve
ER -