Abstract Levodopa, which is usually used in combination with carbidopa, is the gold standard medication in relieving motor symptoms of Parkinson’s disease. There is substantial evidence showing the drugs' stability issues; hence, understanding kinetics and factors affecting drug degradation is of interest. This study highlighted the effects of antioxidants, heat, pH, buffer types, and illumination on the degradation of the two compounds in aqueous solutions. Experimental data showed that levodopa and carbidopa were stable at pH ≤ 5.5 and ≤ 3.0, respectively. Illumination, heat, and neutralized pH accelerated the degradation of the drugs. Carbidopa stability was enhanced when antioxidants (e.g. sodium thiosulfate, disodium ethylenediamine tetraacetic acid) were introduced. The two drugs accelerated the degradation of each other, whereas carbidopa was more susceptible to degradation than levodopa. The presence of levodopa increased carbidopa degradation rate constant by approximately 4 times and 2 times under light shielding and illumination, respectively.

Keywords: Parkinson’s disease, Stability, Levodopa, Carbidopa

Funding: This research was funded by Vingroup Innovation Foundation (VINIF) under project code VINIF.2022.DA00114, Vietnam.

Citation: Dao, N.V., Le, T.T.T., Pham, P.H., Nguyen, P.T., Nguyen, Y.T.H., and Vo, A.Q. 2025.  Impacts of storage conditions and synergistic degradation on the stability of levodopa and carbidopa in aqueous solutions. Natural and Life Sciences Communications. 25(2): e2026031.

Graphical Abstract:

INTRODUCTION

Levodopa (LEV) is the first-line drug in the management of Parkinson’s disease. The compound is a dopamine precursor which can pass the blood-brain barrier. Inside the brain, it is decarboxylated to generate an active ingredient of dopamine which relieves symptoms of the disease. Side effects of the drugs are mainly caused by peripheral decarboxylation which can be minimized by co-treatment of LEV with decarboxylase inhibitors, such as carbidopa (CAR) (Tourtellotte et al., 1980; Samanta and Hauser, 2007; Nyholm et al., 2013). In the combination, CAR inhibits the metabolism pathway of LEV in the circulatory system, that ultimately enhances LEV permeation into the brain. The impact of CAR on LEV kinetics was found to be concentration-dependent (Kaakkola et al., 1985). LEV and CAR are often formulated as tablets, including immediate release tablets, oral disintegrating tablets, controlled release tablets, extendended release capsules, a continuous enteral gel suspension (Livingston and Monroe-Duprey, 2024), and more recently, three dimension-printed tablets for personalized treatment (Langebrake et al., 2023; Rosch et al., 2023). Solutions containing both LEV and CAR can also be prepared from commercial tablets for daily administration by patients with difficulties in swallowing (Vijiaratnam et al., 2017), or patients whose symptoms fail to respond to LEV - CAR tablets (Davis et al., 1993; Kurth et al., 1993).

However, the treatment efficiency of the drugs is affected by their instability and narrow absorption window issues (Klausner et al., 2003; Zhao et al., 2025). Apart from the infusion gel that the formulation is directly pumped into the optimal site of absorption (the duodenum) (Nyholm et al., 2013), other formulations are suspected of pH variation when transited in the gastrointestinal tract that potentially poses significant impacts on drug stability and absorption. Further, owing to aromatic alcohol and hydrazine functional groups (Figure 1), LEV and CAR are prone to oxygen or free radical-initiated oxidative degradation similar to other phenolic compounds (Suat-HianTan and Rezaul Karim, 2018; Guo et al., 2022). Decomposition rate can also be accelerated upon dissolving in aqueous media that is required prior to drug absorption in GI tract, or to sample analysis. The extent of drug stability is of paramount importance, not only in clinical practice (“in use” stability) (Tasana et al., 2024), but also in sample preparation, analysis and other stages of drug development. As such, understanding of degradation kinetics of the LEV and CAR, and any potent impacts of environmental factors, is necessary in the development of better dosage forms containing these active compounds.

Figure 1. Chemical structure of (a) levodopa and (b) carbidopa.

Weitzel group reported the influence of dissolution media (including tap water, whole milk, or simulated gastric fluid) on the stability of LEV - CAR dissolved from combination tablets (Weitzel et al., 2022), and proposed some strategies in patient’s drug administration. The oxidation of LEV was observed to follow first-order kinetics at pH 2.0 and 7.4 (Zhou et al., 2012). LEV’s decay was induced by addition a source of metal (for instance, MgO - a laxative for management of constipation in patient with Parkinson’s disease (Omotani et al., 2016) that ultimately results in reducing drug absorption and treatment efficacy (Pedrosa Carrasco et al., 2018; Miyaue et al., 2022). In another work, LEV was proven to be unstable in the combination solutions and the LEV stability can be improved by temperature alleviation or antioxidant introduction (Pappert et al., 1996). Although phenolic compounds were proved to be fairly stable when handled at 70°C (Nattakan and Patcharee, 2021), it was also shown that CAR degraded in aqueous solutions at a relatively high rate (Pappert et al., 1997), even when protective strategies were applied (such as ambient-light protection or refrigeration storage). The essential role of CAR in maintaining LEV biological activity necessitates comprehensive information regarding the degradation kinetics of CAR solely as well as LEV - CAR combination solution and the impacts of environmental factors on these reactions.

This study aimed to investigate the kinetics of chemical degradation of LEV and CAR depending on pH, buffer ingredients, heat, antioxidants, and illumination. The impact of LEV on CAR’s stability and vice versa was also reported. A comprehensive understanding of the degradation kinetics of LEV and CAR can be useful in dosage form development and clinical practice of levodopa and carbidopa combination drug products.

MATERIALS AND METHODS

Materials

USP-grade levodopa and carbidopa were procured from Zhejiang Wild Wind (China) and Divis’s Laboratories (India), respectively. HPLC-grade acetonitrile and methanol were purchased from Merck KGaA (Germany). Analytical grade boric acid, citric acid monohydrate, hydrochloric acid, disodium ethylenediamine tetraacetic acid dihydrate, potassium dihydro phosphate, sodium acetate, sodium dihydro phosphate dihydrate, sodium hydroxide and sodium thiosulfate were bought from Xilong Scientific Co. Ltd (China). Water was generated by a double-stilled water system (2018 GFL model, Germany).

Methods for simultaneous quantification of levodopa and carbidopa

The HPLC method for LEV and CAR quantification was referred to the Levodopa and Carbidopa Monographs (USP 41) with minor modifications. Briefly, samples were analyzed using an HPLC system (Agilent 1200 Series, Agilent Technologies, Germany, detector DAD- UV VIS) equipped with RP-column C18 (4.6 x 150 mm, 5 μm, GL Sciences, Japan). A phosphate buffer (NaH₂PO₄ 0.05M, pH 2.2) was the mobile phase and was eluted at a flow rate of 2.2 mL/min. A 10 µl sample was injected into the system via a built-in autosampler. Analytical signals were detected at the wavelength 282 nm. Data were finally processed using the ChemStation software (Agilent Techonology Inc., Germany). Prior to analysis, dilute samples containing LEV and/ or CAR in a phosphate buffer (0.1 M, pH 3.0) supplemented with Na₂EDTA (0.01 mg/ mL). The HPLC method was validated in terms of sensitivity, selectivity, linearity, lower limit of quantification, precision and accuracy, and stability, following U.S. FDA guidance for validation of analytical procedures (U.S. Department of Health and Human Services Food and Drug Administration, 2005).

Determination of levodopa - carbidopa degradation in aqueous solutions

General workflow for degradation study

To prepare solutions for the degradation study, dissolve either LEV or CAR or a mixture of LEV and CAR in double distilled water at a predetermined concentration (500 μg/mL for LEV and 125 μg/mL for CAR). The concentration ratio was selected based on a commercial tablet form, Sinemet (Merck, 100 mg LEV, 25 mg CAR). These solutions were transferred into glass containers and were adjusted by adding antioxidants, changing pH or altering buffering agents to identify the impacts of these factors on API chemical stability. Forced degradation assays were performed at elevated temperatures and the obtained kinetics were compared to samples stored at room temperature. Photostability tests were conducted in an illumination cabinet and the results were compared to degradation data in lightproof conditions. At different time points, dilute 100 µl samples by a pH 3.0 buffer prior to LEV and CAR quantification.

Data (concentration versus time) were fit to kinetic equations of simple reactions (i.e. zero, first, and second order kinetics) (Sinko, 2023); reaction orders were determined based on the fitting results via regression coefficients. Kinetic equations of zero, first, and second order reaction were expressed as follows, whereas C_o and C_t are initial concentrations of drugs (t = 0) and at different time points t, respectively; k is the rate constant of degradation:

Impacts of antioxidants and temperature

Solutions containing LEV (500 μg/mL) and CAR (125 μg/mL) were prepared in phosphate buffers (0.05 M) and pH was adjusted to 3.0. To evaluate the chemically stabilizing capacity of antioxidants, Na₂EDTA (10 μg/mL) or Na₂S₂O₃ (0.01 M) was introduced to the solutions and the samples were subsequently stored in lightproof glass vials. All samples were kept at either 55°C or 65°C to spot any effects of storage temperature on drug degradation kinetics. After predetermined time intervals (0; 4; 10; 22; 70; 142 h), concentrations of LEV and CAR were determined and were subsequently compared to levels of the corresponding drug in formulations without antioxidants under the same experimental conditions.

To extrapolate the kinetics of degradation at other temperatures, fit data at accelerated temperatures into the Arrhenius equation formulating temperature dependence of the reaction rate constant (Sinko, 2023).

k₁, k₂, k₃ are degradation rate constants at T₁ = 328 K (55°C), T₂ = 338 K (65°C) and T₃ = 310 K (37°C) or T₃ = 298 K (25°C), respectively; E_A is activation energy (J/mol); R is universal gas constant (8.314 J/mol.K); A is pre-exponential factor.

Impacts of solution pH

Solutions simultaneously containing LEV (500 μg/mL) and CAR (125 μg/mL) were prepared in phosphate buffers (0.05 M NaH₂PO₄, and 10 μg/mL Na₂EDTA). Titrate pH of these solutions to 1.2; 3.0; 4.5; 5.5; 6.8 by using HCl 0.1 N or NaOH 0.1 N. Samples were stored in lightproof glass vials at 65°C, thereafter concentrations of LEV and CAR were determined at various pre-determined time points.

Impacts of buffering agents

LEV (500 μg/mL) and CAR (125 μg/mL) solutions containing Na₂EDTA (10 μg/mL) were prepared at pH 3.0 and the solution pH was maintained by different buffering systems: acetate (0.05 M); citrate (0.05 M); sodium phosphate (0.05 M); potassium phosphate (0.05 M). Samples were stored in amber glass vials at 65°C and concentrations of LEV/CAR were determined at 0; 24; 72; 120; and 168 hours thereafter.

Impacts of illumination

LEV (500 μg/mL) and CAR (125 μg/mL) solutions containing Na₂EDTA (10 μg/mL) were prepared in phosphate buffer (NaH₂PO₄, 0.05 M) at pH 3.0. Samples were transferred into clear glass vials, then placed in an illumination cabinet at room temperature (29 ± 1°C) equipped with a white light source (LED bulb, luminous flux: 2,700 lm, power: 300 W, Yingtan Yankon Lighting Co., Ltd, China). The bulb was kept at 10 cm from testing solutions and each sample vial was light-isolated from the others to avoid any mutual impacts. Light intensity was determined by a digital lux meter (TA8123 series, 0 – 200,000 Lux, repeatability 100 ± 2%, Suzhou Tasi Electronics Co., Ltd, China) and was maintained in the range 16,914 ± 726 Lux. LEV and CAR were quantified at 0; 1; 4; 10; 22; 34; and 46 hours after light exposure.

Mutual impacts on stability of the drugs

Prepare solutions containing only LEV (defined as single solutions) in phosphate buffer pH 3.0 (NaH₂PO₄, 0.05 M) containing Na₂EDTA (10 μg/mL), and the degradation reactions were performed at either temperature stress (65°C) or illumination. The samples were subsequently analyzed to determine LEV concentrations as a function of time. Combination solutions of LEV and CAR were prepared and studied in similar conditions to obtain the degradation profile of LEV in the presence of CAR. To identify any possible impacts associated with CAR presence, the degradation decays of LEV in combination solutions were subsequently compared to that obtained from single solutions.

Similarly, the influence of LEV on CAR degradation kinetics in aqueous media was potentially discerned by comparing CAR degradation profiles in the combination solutions to CAR decomposition decays in single solutions prepared and studied in parallel.

Data analysis

Data processing and statistical analysis were conducted by Microsoft Excel 2022 software and GraphPad-Prism 9.2.0. For each experimental condition, solution samples were prepared in triplicate and analyzed independently. Data were plotted as mean ± standard deviation (SD) from at least three independent experiments.

RESULTS

Impacts of antioxidants and storage temperature on drug degradation

Solutions containing levodopa (LEV) and carbidopa (CAR) were prepared in double distilled water and were subsequently stored at either 55°C or 65°C. At each temperature conditioning, Na₂S₂O₃ or Na₂EDTA was added, and time-dependent levels of LEV - CAR were compared to that of solutions without antioxidants. Table 1 and Figure 2 showed that LEV degraded by less than 5% after 142 hours of storage at both tested temperatures in the presence of Na₂S₂O₃ or Na₂EDTA. Meanwhile, a decrease of 8 - 12 % LEV concentrations was observed in samples without antioxidants introduced under the same temperatures. The data suggested that LEV was more stable with the aid of antioxidants. The effect of temperature on LEV degradation was elucidated by comparing the degradation kinetics of samples without chemically stabilizing agents. LEV was observed to degrade faster at 65°C than at 55°C, however, the change was not significant.

Table 1. Concentrations (% remained) of levodopa and carbidopa in solutions containing the two drugs upon temperature and antioxidant variation (n = 3).

Figure 2. Impacts of storage temperature and antioxidants on the degradation profiles of LEV (a) and CAR (b) in combination solutions (n = 3).

Similarly, the CAR degradation profile was found to depend on solution composition. After 142 hour stored at 55°C, concentrations of CAR upon Na₂S₂O₃ or Na₂EDTA addition, or without antioxidants, dropped to approximately 87, 55, and 14%, respectively. The data for samples stored at 65°C were 82, 18, and 4%, respectively. It was indicated that the employed antioxidants could reduce the decomposition decay of CAR, and Na₂S₂O₃ exhibited better protective ability than Na₂EDTA in this regard. When solutions were supplemented by the same antioxidant, the CAR degradation rate was found to be faster at 65°C than the corresponding rate at 55°C.

Degradation data of CAR (in combination solution) were fitted into simple kinetic equations (reaction order: 0; 1; 2) and the obtained regression coefficient was used for evaluation. When no antioxidant was applied, the degradation data of CAR fitted well with the first-order reaction (R² > 0.96), whereas in the presence of Na₂EDTA, both the zero-order model and the first model could be used (R² > 0.90 and R₂ > 0.92, respectively). With the presence of Na₂S₂O₃, R² value at 55°C was low (0.5 - 0.7), whereas at 65°C, R² fell in the range of 0.81 - 0.87 suggesting in oxygen-free media, CAR decomposition decays follow complicated reactions such as consecutive or parallel pathways or any form of their combination, rather than simple kinetic models. Indeed, as per the discussion below, CAR has a catechol moiety and a hydrazine functional group that are both susceptible to radical-induced and radical-free degradations, and Na₂S₂O₃ has only the capacity to retard former reactions by quenching free oxygen and radicals. It can also be elucidated that in oxygen-dissolved aqueous media, the oxidation of CAR follows simple kinetic models; when oxidation is eliminated (by introducing Na₂S₂O₃), other decomposition pathways are more significant, however, at a slower rate. Nonetheless, a more comprehensive set of data, i.e., with bigger shifts in CAR concentration, should be collected in the future for better kinetic modeling. To simplify the calculation, the first order was selected as the model for CAR decays across all tested conditions. Rate constants of CAR decomposition at 25°C and 37°C were extrapolated from data at elevated temperatures by applying the Arrhenius equation (Table 2). Figure 3 illustrates lnK of CAR kinetics decay as a function of 1/T.   

Table 2. Reaction rate constants of CAR in the presence of LEV at several conditions.

Figure 3. Plot lnK of CAR degradation kinetics as a function of 1/T.

Impacts of pH media on LEV - CAR degradation

Aqueous solutions of levodopa (LEV) and carbidopa (CAR) supplemented with Na₂EDTA were prepared at different pH values and were stored at 65°C. Upon pH variation, both LEV and CAR levels changed accordingly, albeit LEV was found to be more chemically stable than CAR (Figure 4). In the presence of CAR and pH solution varied between 1.2 and 5.5, LEV concentration remained unchanged after 15 h stored at 65°C. However, when the media was the most basic (pH 6.8), LEV decomposition was more pronounced which was demonstrated by an approximate decrease of 24% in LEV levels after 15 h tested.  

The media pH had a greater impact on CAR stability than on LEV stability. In the presence of LEV, change in CAR levels was modest in an acidic environment (pH ≤ 3.0) after 15 h storage at 65°C. In contrast, the higher the pH was, the faster CAR degraded. When the pH solution was 6.8, the reaction rate was highest. CAR concentration dropped to just above 50% after 3 hours, and unsurprisingly, CAR level was not detectable after 11 h assayed at this pH condition. Figure 4 illustrates HPLC traces when analyzed combination solutions at a 6-hour timepoint: while changes in LEV peaks were found modest across four panels, there was a sharp decrease in CAR peaks when the pH solution was increased from 3.0 to 6.8 (Figure 5a to 5d). Such that 3.0 was considered the threshold of environmental pH below which both drugs exhibited enhanced stability and could benefit formular/ clinical practice. The kinetic of decomposition of CAR in combination solutions at 37°C or 25°C (pH 3.0) was extrapolated from elevated tempearture as aformentioned.

Figure 4. Impacts of pH on the degradation profiles of LEV (a) and CAR (b) in combination solutions (n = 3).

Figure 5. Representative chromatograms of LEV and CAR in combination solutions (supplemented with Na₂EDTA at 10 μg/mL) after 6 hour stored at 65°C upon pH variation: (a) pH 3.0; (b) pH 4.5; (c) pH 5.5; (d) pH 6.8.

Impacts of buffering agents on LEV - CAR degradation

Different buffers were used to prepare solutions containing levodopa (LEV) and carbidopa (CAR) and Na₂EDTA. Experimental results exhibited that at pH 3.0, altering buffer types did not significantly affect LEV levels. After 7-day storage at 65°C, concentrations of LEV remained stable across all tested buffering agents (phosphates, citrate, or acetate, Figure 6a).

Figure 6b illustrates concentrations of CAR as a function of time upon buffer alteration at the same pH and temperature conditions. Significantly, CAR was found to degrade completely after 7 days, and changing buffer components neither increased nor decreased CAR degradation. Such that suggested effect of buffering agents on the degradation of both LEV and CAR was negligible and was dominated by the impact of pH media.

Figure 6. Impacts of buffering agents on the degradation profiles of LEV (a) and CAR (b) in combination solutions at pH 3.0 (n = 3).

Impacts of illumination

Solutions containing levodopa (LEV), carbidopa (CAR), and Na₂EDTA were prepared in phosphate buffer (Na₂HPO₄, pH 3.0) and were exposed to a white light source (light iradiance 16,914 ± 726 Lux). After 4-hour illumination, HPLC analysis showed that levels of LEV remained unchanged while CAR concentration dropped to 87% of the initial CAR levels (Figure 7). At the 48-hour time point, approximately 8% of LEV level was reduced, while 95% of CAR concentration was decreased due to photodecomposition, indicating that CAR was highly sensitive to illumination. The data illustrates that LEV and CAR dissolved in aqueous solvents were photo-degraded, and CAR possessed a significantly high reaction rate.

Figure 7. Impacts of illumination on the degradation of LEV - CAR in combination solution (n = 3).

Combination of levodopa - carbidopa favors each drug degradation in aqueous solutions

The impact of carbidopa (CAR) on levodopa (LEV) degradation was elucidated by comparing LEV kinetic profiles in LEV - CAR combination solutions and in solutions containing LEV solely. Figure 8a showed that at temperature stress (65°C), comparable levels of LEV were observed in single solutions and in combination solutions throughout 70 hours (approximately 100% initial concentrations). The data exhibited that degradation profiles of LEV were independent of CAR addition. However, under illuminating conditions (Figure 8b), the decomposition of LEV was augmented in the presence of CAR: after 46 hours of light exposure at irradiance 16,914 ± 726 LUX, LEV in combination solutions decreased by 8% while LEV remained stable in solutions without CAR.

Figure 8. Degradation profiles of LEV and CAR in aqueous solutions containing the two drugs (combination solutions) and containing only one of two drugs (single solution) at different conditions: (a) 65°C, light protected; (b) 29°C, illuminated at irradiance 16,914 ± 726 LUX (n = 3).

Notably, under both temperature stress and illumination, the presence of LEV significantly increased CAR degradation. The impact was evidenced by a greater decrease of CAR concentrations observed from LEV and CAR solutions compared to that from CAR single solution. For instance, after 46 hours of illumination testing, levels of CAR were found to decrease by about 95% and 78%, respectively. Rate constants for CAR degradation, calculated using first-order kinetics, are presented in Table 3 and were further analyzed using a two-way ANOVA model with Tukey’s multiple comparison post-hoc test. The results confirmed that the presence of LEV significantly impacted CAR degradation (P < 0.01), evidenced by a marked increase in the rate constants by approximately 3.5-fold under 65°C with light shielding and by 2-fold under 29°C with illumination. Figure 9 illustrates chromatograms of LEV and CAR in combination solutions after 10 hour illuminated.   

Figure 9. Representative chromatograms of LEV (a) and CAR (b) in combination solutions after 10 hour illuminated at 29°C (irradiance 16,914 ± 726 LUX).

Table 3. Impact of LEV on rate constants of CAR decompositions (n = 3).

DISCUSSION

Both levodopa (LEV) and carbidopa (CAR) contain catechol moieties that can participate in oxidation or radical reactions (Figure 1). The presence of dissolved oxygen can initiate radical formation and, subsequently, the dimerization and polymerization of phenol derivatives (Devlin and Harris, 1984; Saito et al., 2007; Guo et al., 2022). Agents blocking these pathways can exert stabilizing effects on LEV and CAR. Notably, the hydrazine group of CAR makes its stability characteristics distinctive from that of LEV (Figure 8). Hydrazine functional groups (in CAR) are more nucleophilic than amine moieties (in LEV) due to the presence of the adjacent nitrogen. Indeed, hydrazine and its derivatives were shown to be highly reactive towards radicals (Harris et al., 1979) and can also participate in photochemical coupling reactions (Kong et al., 2022). The reactivity of the hydrazine functional group was demonstrated by Nemoto et al. (2003) that even a small amount of oxygen or water can initiate radical formation and chain reactions. Such that the chain reactions led to the decomposition of protected hydrazine derivatives when stored at 5°C under nitrogen, light-protected (Nemoto et al., 2003). The data collected in this study confirmed the discrepancy in LEV - CAR stability that was reported previously (Pappert et al., 1996, 1997; Weitzel et al., 2022). The articles reported that the degradation rate of CAR was markedly accelerated when combination tablets were dissolved in tap water, and no tested strategies provided a protective effect, even under ambient or low-temperature conditions (Pappert et al., 1997; Weitzel et al., 2022). In contrast, our findings demonstrated that when solutions were prepared in double-distilled water at pH ≤ 3.0, CAR stability was significantly enhanced in the presence of either Na₂EDTA or Na₂S₂O₃ (Figure 2, Figure 4), even at an elevated temperature (55°C). These results indicate that pH, media, and antioxidants played critical roles in exerting drug stability of both drugs in solutions. The observed synergistic degradation phenomenon may benefit clinical practice and formulation development.

Roles of media pH and storage temperature

LEV and CAR’s chemical stabilities are dependent on types of dissolution media (Weitzel et al., 2022). Both drugs were stable in simulated gastric fluid (pH 1.2), however, CAR was less stable than LEV when dissolved in tap water or in whole milk. In line with this finding, our data herein demonstrated that two drugs exhibited pH-dependent stability profiles, and CAR was more susceptible to changes in pH media. LEV was found to be stable at pH ≤ 5.5 and the corresponding threshold for CAR was 3.0. In order to explain this discrepancy, pK_a of catechol functional groups should be taken into consideration (Lin et al., 2022). The first pK_a value was found to vary in the range of 6.6 - 9.6 depending on substituted groups of the benzyl ring, while the second pK_a was shown to be comparatively higher (Romero et al., 2018). It can be inferred that at pH media ≤ 5.5 < pK_a1, most -OH functional groups in the catechol moieties exist in an unassociated form that unfavor oxidation or electrophile addition to the ring. That said, the pH susceptibility of CAR could be attributed to the pH-dependent reactivity of the -NH-NH₂ functional group (Nakui et al., 2007). The proximal small intestine (the duodenum), the optimal site of absorption for both LEV and CAR (Klausner et al., 2003; Samanta and Hauser, 2007), possesses a pH value of approximately 6.6 that might favor drug degradation (Evans et al., 1988; Omotani et al., 2016; Miyaue et al., 2022). However it can be postulated that the degradation is modest due to the short transit time along the duodenum (Davis et al., 1993). Nonetheless, in liquid samples containing LEV and CAR (Metman et al., 1994; Samanta and Hauser, 2007), the environmental pH should be adjusted to below 3.0 to optimize drug stability. This strategy is clinically practical, as numerous fruit juices exhibit pH values near 3.0 (Reddy et al., 2016) that can be used for dispersing combination tablets. Furthermore, storage at reduced temperatures (e.g., refrigeration) is strongly recommended to mitigate the degradation of liquid formulations, particularly under conditions where the environmental pH exceeds 3.0.

Antioxidant mechanisms

When combination solutions were prepared at pH 3.0 and kept in lightproof containers, experiments showed that Na₂S₂O₃ exhibited better protecting ability on CAR than Na₂EDTA did. This effect probably stemmed from their distinct working mechanisms. Na₂EDTA acts as a chelate complex agent that can block ion-induced catalytic reactions. Meanwhile, Na₂S₂O₃ is a strong reductant, reacts directly with dissolved oxygen and diminishes radical formation. The compound is able to retard both metal-catalytic and non-catalytic oxidative reactions of CAR, thereby exerting better stabilizing effects. However, other reaction pathways might be induced during the course of temperature stress, including heat degradation and amid formation (between -NH-NH₂ and -COOH functional groups) (Bhardwaj et al., 2023). Antioxidants are unlikely to stabilize CAR in this situation. This finding was in line with several published work (Pappert et al., 1997; Miyaue et al., 2022), ascorbate (a reductant and antioxidant) was able to alleviate the CAR degradation rate in a concentration-dependent manner rather than diminishing the reaction. A combination of multiple antioxidant strategies (such as metal chelation, oxygen scavenging, and radical quenching) is recommended for the handling of liquid formulations containing LEV and/or CAR to exert drug stability.

Illumination and effects and synergistic degradation

Illumination posed significant impacts on LEV and CAR degradation by propagating radical formation and chain reaction. Owing to two sites of radical reactions (catechol and hydrazine groups), CAR was found to be more prone to light shedding. Notably, the presence of LEV significantly enhances CAR decomposition upon light exposure and versa. This effect possibly stemmed from the fact that under this condition, each drug reacts with radicals and becomes a secondary source of radicals that participate in chain reactions. Indeed, the initial concentration of LEV was 4 times higher than CAR, and CAR possesses two reactive functional moieties. Such that the impact of LEV on CAR degradation was more significant than the impact of CAR on LEV decays. The data suggest that when proper stabilizing approaches are applied, changes in LEV and CAR levels are minimized. For instance, ascorbic acid introduction was able to alleviate metal-induced decomposition that might happen in gastrointestinal tracts, thereby improving LEV - CAR pharmacokinetics (Kashihara et al., 2019; Miyaue et al., 2022). We also conducted an experiment that mimicked dissolution tests (data not shown), LEV and CAR concentrations were found to remain unchanged when either single solutions or combination solutions (pH 3, supplemented with Na₂S₂O₃ at 0.01 mg/mL) were continuously stirred (100 rpm) for 24 hours in lightproof containers. Isolation drug by formulating LEV - CAR resin was proven to greatly reduce drug degradation, as discussed in Liu’s work (Liu et al., 2018). Where applicable, reduced intensity light sources (i.e. yellow light with low irradiance) should be considered when handling with LEV - CAR solutions.

Although the roles of pH, buffer components, temperature, antioxidants, illumination, and synergistic degradation in LEV and CAR decomposition have been examined and clarified, several additional experiments remain of interest for future investigation. For instance, the mutual impacts of the drugs in solid state associated with tablet compression, powder compounding, along with matrix ingredients are not yet understood. Furthermore, the implications of simulated gastric or intestinal fluid (fasted and fed conditions) are also important, given that the presence of endogenous antioxidants (ascorbic acid, glutathione…) and other biological molecules may induce or retard degradation reactions.

Overall, the data in this study suggest that heavy ions and dissolved oxygen should be eliminated in practical experiments of solutions containing LEV and CAR. Drug solutions, specifically CAR solutions, should be avoided from light sources and high temperatures to minimize drug decomposition. Where applicable, antioxidants should be introduced, and samples containing these drugs should be acidified to pH ≤ 3.0 to retard the degradation process. Besides, combination solutions of LEV and CAR should be prepared right before use. Data in this study are beneficial not only to clinical practice but also to stability testing and formulation development of the drugs.

CONCLUSION

Levodopa (LEV) was stable at pH ≤ 5.5 and its stability was modestly impacted by buffers, temperature alleviation, illumination, antioxidants and the presence of carbidopa (CAR). Carbidopa was stable at pH ≤ 3.0 and the degradation was significantly induced at high pH or upon accelerated temperature or light exposure. The addition of LEV increased the CAR degradation rates. The current study provides valuable information that can benefit dosage form development, quality control, and clinical practice of drugs containing LEV and CAR.

ACKNOWLEDGEMENTS

The authors thank the Faculty of Pharmaceutics and Pharmaceutical Technology, Hanoi University of Pharmacy for providing instruments.

AUTHOR CONTRIBUTIONS

Nam V. Dao: Experimental Design (Lead), Methodology (Lead), Performing Experiments (Support), Data Analysis (Lead), Writing Original Draft & Revision (Lead); Trang T.T. Le: Performing Experiments (Support), Data Analysis (Equal); Phuc H. Pham: Performing Experiments (Equal), Data Collection & Process (Support); Phuc T. Nguyen: Performing Experiments (Equal), Data Collection & Process (Support); Yen T.H. Nguyen: Performing Experiment (Equal), Data Collection and Process (Support); Anh Q. Vo: Conceptualisation (Lead), Funding Acquisition (Lead), Project Administration (Lead), Resources (Lead), Supervision (Lead), Data Interpretation (Support), Manuscript Review & Editing (Lead). All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

REFERENCES

Bhardwaj, N., Tripathi, N., Kumar, S., and Jain, S.K. 2023. Synthesis of amides directly from carboxylic acids and hydrazines. Organic & Biomolecular Chemistry. 21(37): 7572-7579. https://doi.org/10.1039/D3OB01268A

Davis, S.S., Wilding, E.A., and Wilding, I.R. 1993. Gastrointestinal transit of a matrix tablet formulation: Comparison of canine and human data. International Journal of Pharmaceutics. 94(1): 235-238. https://doi.org/10.1016/0378-5173(93)90029-F

Devlin, H.R. and Harris, I.J. 1984. Mechanism of the oxidation of aqueous phenol with dissolved oxygen. Industrial & Engineering Chemistry Fundamentals. 23(4): 387-392. https://doi.org/10.1021/i100016a002

Evans, D.F., Pye, G., Bramley, R., Clark, A.G., Dyson, T.J., and Hardcastle, J.D. 1988. Measurement of gastrointestinal pH profiles in normal ambulant human subjects. Gut. 29(8): 1035-1041. https://doi.org/10.1136/gut.29.8.1035

Guo, B., Chou, F., Huang, L., Yin, F., Fang, J., Wang, J.-B., and Jia, Z. 2022. Recent insights into oxidative metabolism of quercetin: Catabolic profiles, degradation pathways, catalyzing metalloenzymes and molecular mechanisms. Critical Reviews in Food Science and Nutrition. 64(5): 1312-1339. https://doi.org/10.1080/10408398.2022.2115456

Harris, G.W., Atkinson, R., and Pitts, J.N. 1979. Kinetics of the reactions of the hydroxyl radical with hydrazine and methylhydrazine. The Journal of Physical Chemistry. 83(20): 2557-2559. https://doi.org/10.1021/j100483a001

Kaakkola, S., Männistö, P.T., Nissinen, E., Vuorela, A., and Mäntylä, R. 1985. The effect of an increased ratio of carbidopa to levodopa on the pharmacokinetics of levodopa. Acta Neurologica Scandinavica. 72(4): 385-391. https://doi.org/10.1111/j.1600-0404.1985.tb00888.x

Kashihara, Y., Terao, Y., Yoda, K., Hirota, T., Kubota, T., Kimura, M., Matsuki, S., Hirakawa, M., Irie, S., and Ieiri, I. 2019. Effects of magnesium oxide on pharmacokinetics of L-dopa/carbidopa and assessment of pharmacodynamic changes by a model-based simulation. European Journal of Clinical Pharmacology. 75(3): 351-361. https://doi.org/10.1007/s00228-018-2568-4

Klausner, E.A., Eyal, S., Lavy, E., Friedman, M., and Hoffman, A. 2003. Novel levodopa gastroretentive dosage form: In-vivo evaluation in dogs. Journal of Controlled Release. 88(1): 117-126. https://doi.org/10.1016/S0168-3659(02)00487-X

Kong, Y., Wei, K., and Yan, G. 2022. Radical coupling reactions of hydrazines via photochemical and electrochemical strategies. Organic Chemistry Frontiers. 9(21): 6114-6128. https://doi.org/10.1039/D2QO01348G

Kurth, M.C., Tetrud, J.W., Irwin, I., Lyness, W.H., and Langston, J.W. 1993. Oral levodopa/carbidopa solution versus tablets in Parkinson's patients with severe fluctuations: A pilot study. Neurology. 43(5): 1036-1039. https://doi.org/10.1212/WNL.43.5.1036

Langebrake, C., Gottfried, K., Dadkhah, A., Eggert, J., Gutowski, T., Moritz, R., Nils, S., Christopher, G., Sylvia, N., Frank, Ü., et al. 2023. Patient-individual 3D-printing of drugs within a machine-learning-assisted closed-loop medication management - Design and first results of a feasibility study. Clinical eHealth. 6: 3-9. https://doi.org/10.1016/j.ceh.2023.05.001

Lin, S., Zhang, H., Simal-Gandara, J., Cheng, K.-W., Wang, M., Cao, H., and Xiao, J. 2022. Investigation of new products of quercetin formed in boiling water via UPLC-Q-TOF-MS-MS analysis. Food Chemistry. 386: 132747. https://doi.org/10.1016/j.foodchem.2022.132747

Liu, H., Ding, H., Zhang, D., Fengsi, and Sun, C. 2018. Report: Preparation of levodopa/carbidopa compound drug resins. Pakistan Journal of Pharmaceutical Sciences. 31(1): 205-211.

Livingston, C. and Monroe-Duprey, L. 2024. A review of levodopa formulations for the treatment of Parkinson's disease available in the United States. Journal of Pharmacy Practice. 37(2): 485-494. https://doi.org/10.1177/08971900221151194

Metman, L.V., Hoff, J., Mouradian, M.M., and Chase, T.N. 1994. Fluctuations in plasma levodopa and motor responses with liquid and tablet levodopa/carbidopa. Movement Disorders. 9(4): 463-465. https://doi.org/10.1002/mds.870090416

Miyaue, N., Kubo, M., and Nagai, M. 2022. Ascorbic acid can alleviate the degradation of levodopa and carbidopa induced by magnesium oxide. Brain and Behavior. 12(7): e2672. https://doi.org/10.1002/brb3.2672 

Nakui, H., Okitsu, K., Maeda, Y., and Nishimura, R. 2007. The effect of pH on sonochemical degradation of hydrazine. Ultrasonics Sonochemistry. 14(5): 627-632. https://doi.org/10.1016/j.ultsonch.2006.11.008

Nattakan, J. and Patcharee, K. 2021. Effect of foam-mat drying conditions on antioxidant activity, total phenolic compound, anthocyanin content and color of purple-fleshed sweet potato powder. Chiang Mai University Journal of Natural Science. 20(2): e2021045. https://doi.org/10.12982/CMUJNS.2021.045

Nemoto, T., Lazoura, E., and Nomoto, T. 2003. Characterization of hydrazine derivative: Proposed decomposition mechanism and structure elucidation of decomposition compounds. Chemical and Pharmaceutical Bulletin (Tokyo). 51(3): 346-350. https://doi.org/10.1248/cpb.51.346

Nyholm, D., Odin, P., Johansson, A., Chatamra, K., Locke, C., Dutta, S., and Othman, A.A. 2013. Pharmacokinetics of levodopa, carbidopa, and 3-O-methyldopa following 16-hour jejunal infusion of levodopa-carbidopa intestinal gel in advanced Parkinson's disease patients. The American Association of Pharmaceutical Scientists Journal. 15(2): 316-323. https://doi.org/10.1208/s12248-012-9439-1

Omotani, H., Yasuda, M., Ishii, R., Ikarashi, T., Fukuuchi, T., Yamaoka, N., Mawatari, K., Kaneko, K., and Nakagomi, K. 2016. Analysis of l-DOPA-derived melanin and a novel degradation product formed under alkaline conditions. Journal of Pharmaceutical and Biomedical Analysis. 125: 22-26. https://doi.org/10.1016/j.jpba.2016.03.019

Pappert, E.J., Buhrfiend, C., Lipton, J.W., Carvey, P.M., Stebbins, G.T., and Goetz, C.G. 1996. Levodopa stability in solution: Time course, environmental effects, and practical recommendations for clinical use. Movement Disorders. 11(1): 24-26. https://doi.org/10.1002/mds.870110106

Pappert, E.J., Lipton, J.W., Goetz, C.G., Ling, Z.D., Stebbins, G.T., and Carvey, P.M. 1997. The stability of carbidopa in solution. Movement Disorders. 12(4): 608-610. https://doi.org/10.1002/mds.870120422

Pedrosa Carrasco, A.J., Timmermann, L., and Pedrosa, D.J. 2018. Management of constipation in patients with Parkinson's disease. NPJ Parkinson's Disease. 4: 6. https://doi.org/10.1038/s41531-018-0042-8

Romero, R., Salgado, P.R., Soto, C., Contreras, D., and Melin, V. 2018. An experimental validated computational method for pKa determination of substituted 1,2-dihydroxybenzenes. Frontiers in Chemistry. 6: 208. https://doi.org/10.3389/fchem.2018.00208

Rosch, M., Gutowski, T., Baehr, M., Eggert, J., Gottfried, K., Gundler, C., Nürnberg, S., Langebrake, C., and Dadkhah, A. 2023. Development of an immediate release excipient composition for 3D printing via direct powder extrusion in a hospital. International Journal of Pharmaceutics. 643: 123218. https://doi.org/10.1016/j.ijpharm.2023.123218

Saito, K., Sun, G., and Nishide, H. 2007. Green synthesis of soluble polyphenol: Oxidative polymerization of phenol in water. Green Chemistry Letters and Reviews. 1(1): 47-51. https://doi.org/10.1080/17518250701756975

Samanta, J. and Hauser, R.A. 2007. Duodenal levodopa infusion for the treatment of Parkinson's disease. Expert Opinion on Pharmacotherapy. 8(5): 657-664. https://doi.org/10.1517/14656566.8.5.657

Sinko, P.J. 2023. Martin's physical pharmacy and pharmaceutical sciences. Wolters Kluwer- Lippincott Williams & Wilkins, Philadelphia.

Suat-Hian T. and Karim, M.R. 2018. Effects of three drying treatments on the polyphenol content, antioxidant and antimicrobial properties of Syzygium aromaticum extract. Chiang Mai Journal of Science. 45(2): 937-948.

Tasana, P., Kitiya, K., and Natthareephon, P. 2024. Development and stability study of oseltamivir reconstitutable dry suspension from capsules. Natural and Life Sciences Communications. 23(1): e2024010. https://doi.org/10.12982/NLSC.2024.010

Tourtellotte, W.W., Syndulko, K., Potvin, A.R., Hirsch, S.B., and Potvin, J.H. 1980. Increased ratio of carbidopa to levodopa in treatment of Parkinson's disease. Archives of Neurology. 37(11): 723-6. https://doi.org/10.1001/archneur.1980.00500600071015

U.S. Department of Health and Human Services Food and Drug Administration 2005. Q2(R1) validation of analytical procedures: Text and methodology. Guidance for Industry.

Vijiaratnam, N., Cheng, S., Bertram, K.L., and Williams, D.R. 2017. Liquid levodopa/carbidopa: Old solution, forgotten complication. Journal of Movement Disorders. 10(3): 164-165. https://doi.org/10.14802/jmd.17024

Weitzel, J., Wünsch, A., Rose, O., and Langer, K. 2022. Different dissolution conditions affect stability and dissolution profiles of bioequivalent levodopa-containing oral dosage forms. International Journal of Pharmaceutics. 629: 122401. https://doi.org/10.1016/j.ijpharm.2022.122401

Zhao, X., Yan, P., Zhang, H., Zhou, W., and Ding, J. 2025. A novel levodopa-carbidopa three-layer gastroretentive tablet for improving levodopa pharmacokinetics. European Journal of Pharmaceutics and Biopharmaceutics. 207: 114633. https://doi.org/10.1016/j.ejpb.2025.114633

Zhou, Y.Z., Alany, R.G., Chuang, V., and Wen, J. 2012. Studies of the rate constant of L-DOPA oxidation and decarboxylation by HPLC. Chromatographia. 75(11): 597-606. https://doi.org/10.1007/s10337-012-2229-1

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