A New Approach for Studying the Stability and Degradation Products of Ascorbic acid in Solutions

The potentiometric titration (PT) method has been applied for the first time to investigate the stability of l-ascorbic acid (H2Asc) and to determine its degradation products in aqueous solutions. The presented electrochemical procedures can be considered to be a fast, simple and inexpensive way to control the stability of H2Asc in a regular analytical practice as well as in the chemical and pharmaceutical industries. Experimental data as well as modeling suggest that the enolic form of 2,3-diketogulonic acid predominates in a solution as the main product of dehydroascorbate degradation. Furthermore, the PT results supported by conductometric measurements and electrospray ionization mass spectrometry data enable us to propose the putative mechanism of the H2Asc decomposition. Moreover, it has been proven that among different types of investigated electrolytes (KNO3, KClO4 and KSCN), the thiocyanate ions (SCN−) reveal the stabilizing effect against the degradation of H2Asc. Thus, the presence of SCN− in the H2Asc solution is proposed as an alternative way for some organic solvents earlier used. Finally, a new paraffin-protection-layer procedure has been recommended for studying as well as storage of the solutions comprising components sensitive to external factors (e.g. O2, CO2) and to evaporation.


Introduction
l-ascorbic acid (known as vitamin C) is a water-soluble molecule commonly present in plants (fruits and vegetables) [1]. However, humans are not able to synthesize this acid endogenously [2,3]. On account of the well-documented health benefits, vitamin C is widely used in food, cosmetics and pharmaceutical industries. The acid and ascorbates are involved in many biochemical processes that have a proven protective potential against diseases of various etiology including cancer, cardiovascular or diabetes [4]. Furthermore, due to its high reactivity towards reactive oxygen and nitrogen species (RONS) [5], ascorbate 1 3 is considered to be an outstanding antioxidant, co-antioxidant (enhancing the antioxidant action of vitamin E in lipid peroxidation). On the other hand, ascorbate can also act as a pro-oxidant, capable of the catalytic reduction of some biologically important metal ions such as Fe 3+ to Fe 2+ . These redox processes are found to contribute indirectly to the oxidation of the single state organic molecules [6,7].
From the acid-base equilibria point of view, in aqueous solutions ascorbic acid behaves as diprotic acid (denoted as H 2 Asc), namely a keto lactone form with two ionizable OH groups: pKa 1 = 4.2 and pKa 2 = 11.6 [1,8]. In aqueous solutions with pH < pK a1 ascorbic acid exists mainly in a non-ionized form (H 2 Asc). At a physiological pH (7.4) ascorbate species, HAsc − , dominate (99.95%), whereas the content of Asc 2− is negligibly small. This suggests that analyzing the stability of vitamin C in water both forms, i.e. H 2 Asc and HAsc − should be considered. The pH of a solution affects the types of the ascorbic acid species as well as their concentration ( Fig. 1. Increase in the pH of a solution results in a decrease in the stability of HAsc − ions. Under physiological conditions, the HAsc − ions are recognized as reducing agents and readily undergo two-electron oxidation and deprotonation process leading through ascorbyl free radical HAsc • , ascorbate free radical (Asc •− ) to dehydroascorbic acid (DHA) (Fig. 1). Moreover, the formation of DHA is also possible in a highly basic environment through two-electron oxidation process of Asc 2− anions [9]. Redox processes of ascorbic acid species are light and temperature-sensitive and also depend on other factors, among which the presence of oxygen and metal ions plays a crucial role.
DHA is a neutral compound with a lactone structure and a carbonyl moiety (3-C=O) that makes this structure very sensitive to different types of reagents: nucleophiles, oxidants and others. DHA can be reduced to H 2 Asc or irreversible hydrolysed to 2,3-DKGA (2,3-diketogulonic acid) which loses biological activity. Interconversion between H 2 Asc and DHA via radical (HAsc. − ) has been the subject of interest of many research groups Fig. 1 Acid-base and electrochemical equilibria of ascorbic acid in an aqueous solution 1 3 [10,11]. However, there is still little evidence that this phenomenon could take place in vivo [12].
In general, the stability of ascorbic acid solutions depends on the composition of a system, namely the presence of catalytic metals ions, the type of inorganic anions, a pH of a solution as well as storage method. These factors affect the quality of a stock solution of ascorbic acid and thus the reproducibility of the results. It has been reported that most of the degradation products of H 2 Asc are organic acids (carboxylic and OH-acids) with the general formula: H n L (where n denotes the number of protons undergoing dissociation: n = 1, 2, …). They were identified by ESI/MS method as 2,3-diketogulonic acid (2,3-DKGA), threnoic acid (ThA) and oxalic acid (OxA) Fig. 2.
In contrast to these previous studies, in this paper the potentiometric titration method supported by conductometric measurements and electrospray ionization mass spectrometry (ESI/MS) has been employed for the first time to describe H 2 Asc degradation products in aqueous solutions. In addition, we have focused our attention on the stabilizing effect of thiocyanate on ascorbic acid in aqueous solutions. Based on experimental data a putative mechanism of thiocyanate action has been proposed.

The Application of Potentiometric Titration Technique for Studying Degradation Products of l-Ascorbic Acid-A Theory
Potentiometric titration supplemented by theoretical calculations (simulations of titration curves) has been proposed as a useful tool for studying the stability of l-ascorbic acid in an aqueous solution as well as for a quantified assessment of its degradation products.
In the system under study, l-ascorbic acid (H 2 Asc) and acidic species formed as a result of the H 2 Asc degradation, namely 2,3-diketogulonic acid (HA), threnoic acid (HB) and oxalic acid (H 2 C) are presented. All these species have been taken into consideration during the analysis of the composition of a mixture of l-ascorbic acid (H 2 Asc) (Fig. 2). The acid dissociation constants of the components are available in the literature and were used in calculations. The individual equilibria for all species in the mixture are presented in (Table 1).

CerkoLab System EQSOL Software Description
CerkoLab EQSOL (EQSOL) program runs on a Windows operating system and has a user friendly graphics interface similar to other CerkoLab System products [22]. The implemented numerical procedure: EQSOL is based on a revised by Liwo cvequid algorithm earlier presented by Kostrowicki and Liwo [23,24].
The equilibrium state can be described in terms of an ideal solution model, Debye-Hüeckel or Davies models. Based on a model, EQSOL builds a stoichiometric matrix which is subsequently used in numerical procedures.
Sample parameters are fitted to the model by minimalization of a sum of squares of a target function [24]. EQSOL algorithm allows for all, or specific model parameters to be evaluated. The result of the minimalization procedure is in a form of a listing of evaluated parameters and a plot of fitted titration curves. In addition to fitting titration parameters, EQSOL can present plots of equilibrium concentrations, activity coefficients and ionic strength. Additionally, the EQSOL simulation module can be used to predict the titration curve for a specific concentration of reagents and their pK a values.

Titration Curves Simulation
The titration curves for H 2 Asc titration with 0.1 mol·L -1 NaOH (C T ) simulated in the EQSOL are shown in Fig. 3. Calculations were performed for two models: Model ( At the pH ranging from 1 to 9, the shape of the PT simulated curves for both employed models are the same (Fig. 3). However, above a pH of 9, the shape of the PT curves differ Additionally, the titration curves for the above two systems with different types of possible degradation products (HA, HB and H 2 C) have been simulated with the EQSOL software (Fig. 4). The equilibrium models used in the theoretical simulations are listed below. DHA is coming from approx. 2% degradation of H 2 Asc. pK a values for HA = 2,3-diketogulonic acid, HB = threnoic acid, H 2 C = oxalic acid are taken from Table 1.

Materials
l-Ascorbic acid, H 2 Asc, (analytical grade) was purchased from Chempur. Potassium salts of chloride, nitrate, perchlorate and thiocyanate (> 99.5%) were supplied by Sigma-Aldrich, Poland. Sodium hydroxide solution as titrant was prepared from fixanal delivered by Avantor Performance Materials Poland S.A. All reagents were used as obtained without further purification. Deionized water with conductivity not exceeding 0.18 µS·cm − 1 (a HydroLab water purification system) was used for preparation of aqueous solutions.

Preparation of Solution and Incubation Conditions
The ascorbic acid solution C o = 0.010 (± 0.002) mol·L -1 was prepared by dissolving an appropriate amount of analytical grade H 2 Asc in distilled water or in a 0.0050 (± 0.0002) mol·L -1 solution of electrolyte (KNO 3 , KClO 4 and KSCN). Prepared solutions were stored in 0.025 L volumetric flasks at laboratory temperature for 0, 3, 6, 24, 48 and 72 h.

Titration Experiments
Titration experiments were performed in a 30-mL thermostated (298.15 ± 0.1 K) cell using the Cerko Lab System microtitration unit fitted with the 5-mL Hamilton's syringe. The temperature of measured solutions was controlled by circulating water through a jacketed beaker (Julabo-25 circulation thermostat).

Potentiometric Titration Procedure
The titrant (T) was added to the titrand (D) in increments of 0.01 or 0.005 L·10 − 3 , with a pause of 20 s. Each titration was repeated a least twice to check the reproducibility of the data.

Conductometric Measurements (CM)
Conductometric measurements were accomplished immediately after a solution preparation on the CerkoLab System -kinetic unit in a measuring cell equipped with a

Paraffin-Protection-Layer Procedure
To protect the titrand solution from the components present in the laboratory air (especially CO 2 and O 2 ) and to eliminate evaporation of a solvent (here H 2 O) a special procedure was developed. On the top of the titrand (D) a 0.5 cm thick layer of paraffin was deposited from a syringe fitted with a Teflon tube. The conductivity measurement was registered by CerkoLab System as described in the standard procedure.

Validation of Conductometric Procedure Data
The linearity test of a conductometric cell was performed using a standard solution of KCl Additionally, we have compared the conductometric data obtained according to the standard procedure and the proposed paraffin-protection-layer procedure (Fig. 6). It has been proven that the paraffin layer effectively protects the titrand (D) from external environmental conditions and thus guarantees the reproducibility of the data irrespective of the measurement time.

Electrospray Ionization Mass Spectrometry (ESI/MS) Measurements
Agilent liquid chromatograph series 1290 (Agilent Technology, Santa Clara, CA, USA) with quaternary pump G4204A, autosampler G4226A, thermostated column compartment G1316C, and triple quadrupole mass spectrometer G6460C with AJS electrospray ionization source was used for flow injection analysis. The chromatographic system was controlled with Agilent MassHunter software B 06.01. Samples (5 µL) were directly injected. The mobile phase flow rate was 0.2 mL·min -1 , and elution was carried out in water with 10 mmol·L −1 ammonium formate. All mass-spectrometric scan data were collected in negative ionization scan mode at 45 psi nebulizer pressure, 5 L·min -1 nitrogen flow, 573.15 Knitrogen temperature, 11 L·min -1 sheath gas (nitrogen) flow, and 623.15 K sheath gas temperature. The fragmentation voltage was 100 V and the capillary voltage was 3.5 kV. The collision cell radio 1 3 frequency voltage was deactivated, the first quadrupole was in total transmission ion mode, and the second quadrupole was scanning, resulting in a scan mode. The fragmentor voltage controlled the rate at which ions pass through a medium pressure zone and are fragmented by collisions with nitrogen molecules in this mode.

Analysis of pH-metric Titration Data-Numerical and Modeling Procedure
The numerical procedure described before [22] for pK a calculation, the composition of titrand D and the modeling of titration curves is based on the EQSOL.

Potentiometric Titration Analysis (PT)
The potentiometric titration method was applied for qualitative investigations of degradation products of ascorbic acid in aqueous solutions in the presence and the absence of some potassium salts, namely KNO 3 , KClO 4 and KSCN (Hofmeister series). The representative titration curves of ascorbic acid solutions prepared immediately before the titrations as well as after the incubation time at room temperature protected against CO 2 and O 2 present in a laboratory atmosphere for 24-48 h are collected in Fig. 7. Shape of pH titration curves The composition of examined ascorbic acid solutions was assessed based on the differences in the acid-base properties (their pK a values) of the resulting degradation species. The general procedure elaborated previously for the pK a 's determination in a mixture of compounds has been applied [22]. The calculated pK a 's values were subsequently assigned to the functional groups of particular species (Table 2). *pK a3 -COOH, pK a4 -OH (enol form), Fig. 8. Calculated dissociation constants of H 2 Asc (pK a1 and pK a2 ) stay in line with literature values ( Table 1). The oxidation of H 2 Asc leads to the formation of acidic products. This manifests itself in the change of the shape of the pH curves. Fitting of calculated curves to experimental data reveals some additional equilibrium reactions denoted as pK a3 and pK a4 which can be assigned to either diprotic species of the type H 2 A or to two monoprotonated species HA + HB ( Table 1). The EQSOL algorithm adopted in the CerkoLab software has been employed to calculate theoretical potentiometric titration curves for two systems: H 2 Asc + H 2 A and H 2 Asc + HA + HB + H 2 C, Fig. 9. Obtained theoretical results have subsequently been compared with the experimental data. It has been found that the oxidation of H 2 Asc leads to the formation of H 2 A species which fits well to 2,3diketogulonic acid (2,3-DKGA) present in a solution as an enol form (2,3-DKGA) with pKa 3 and pK a4 values close to 3 (-COOH) and 9 (-OH enol form)respectively.
Theoretical titration curves were generated from CerkoLab EQSOL using pK a data for H 2 Asc, HB and H 2 C as in (Table 1) and pK a3 = 3.0 and pK a4 = 9 for H 2 A = 2,3-DKGA. Experimentally determined pK a values for 2,3-DKGA as a degradation product of H 2 Asc in water solution and in the presence of electrolyte are presented in (Table 2). Potentiometric titration data revealed that the thiocyanate ions effectively protect the ascorbate species (HAsc -) against oxidation. On the other hand, the protection action has not been observed for KNO 3 and KClO 4 (Fig. 10). The shape of titration curves of  (Table 2). This proves the presence of the same oxidative degradation products. The only differences are found in the concentration of particular components of titrand D. Furthermore, the concentration of ascorbic acid gradually decreases in time with the increase of the 2,3-DKGA (enol form) concentration ( Table 3). The HAsc − ion is a highly effective reducing agent which easily undergoes oneelectron oxidation to ascorbate radical (HAsc · ) and then to dehydroascorbic acid (DHA) according to the following electron transfer process:  A surprising protective action of thiocyanates remains to be elucidated. Nevertheless, it can be supposed that the putative mechanism of action of SCN − ions in the protection of the ascorbic acid against oxidation involves the formation of the SCN · radicals which participate in the scavenging of ascorbate free radical (Asc · ), the precursor of DHA: SCN − + HAsc · ⇌ HAsc -+ ·SCN · step (1). Overall the reaction. HAsc · + Asc ·− ⇌ · HAsc -+ Asc 2-. The reaction of thiocyanate ion (SCN − ) with HAsc· is proposed by analogy to the reaction of SCN − with other radicals for example with (OH · ), [25]. Moreover, SCN · can react with the excess of SCN − resulting in the formation of the (SCN) 2 · radical [26,27].

Protective Action of KSCN and a Paraffin Layer Against the L-Ascorbic Acid Degradation in an Aqueous Solution -Conductometric Measurements (CM)
The oxidation of H 2 Asc to dehydroascorbic acid (DHA, approx. 1.2%) initially manifests itself in the decrease in the conductivity of a solution. After a time, the conductivity increases slightly, Fig. 11 which is due to the DHA ring opening reaction leading to the formation of 2,3-DKGA, Fig. 8. This phenomenon stays in line with the results of potentiometric titration of H 2 Asc samples prepared immediately before the experiments and after storage of solutions for 6 h in the volumetric flask at laboratory temperature, Fig. 12.
The same phenomena were observed in conductometric curves obtained for mixtures of H 2 Asc and 0.005 M KNO 3 or KClO 4 stored for 24 h under the same conditions. On the other hand, a protective effect against the degradation has been found for the H 2 Asc solution stored under the paraffin layer in the presence of KSCN (Fig. 13). Taking in mind the influence of gaseous species (O 2 and CO 2 ) and also evaporation of the solvent (H 2 O) we have elaborated a special procedure for conductometric measurements based on a 100% protection of a solution against above mentioned physical and chemical factors. When the experiments were carried out under the paraffin layer (see exp. section) we observed a stable value of conductivity for KSCN solution (κ = 917 µS·cm − 1 ) and an increased value of conductivity for aqueous solution, Figs. 11A and B and 13. It is a new procedure recommended for different types of potentiometric and conductometric measurements (titrations as well as kinetic studies conducted for a long period of time) and especially for O 2 , CO 2 sensitive systems. 1 3

The ESI(-)/MS Analysis
The putative mechanism of the H 2 Asc decomposition presented in this study is in good agreement with the chemistry of ketolactone and α-keto carboxylic acids. The first step of the degradation of H 2 Asc is assigned to the oxidation of H 2 Asc to DHA. The next steps of chemical transformations suggested by different authors are shown in Fig. 8. However, the decomposition pathways of H 2 Asc depend on different factors and are still the subject of discussion by many research teams. A few important discrepancies in the nomenclature as well as in the structure of the degradation products proposed mainly based on ESI/MS analysis are found in literature. Among these, a lot of discussion and controversy can be found in [28] (Table 4).  Observed stabilization effect of the thiocyanate ion on H 2 Asc is in good agreement with potentiometric and conductometric results. Additionally, we have observed the peak with m/z = 272 that confirms the cluster formation of the ascorbyl ion with structure: [HAsc − SCN − ]K + (Table 5). According to our knowledge, the formation of clusters of the type [HAsc − A − ]K + (A − denotes SCN − , NO 3 − , ClO 4 − , SCN − ) has been proven for the first time in this paper. The analytical importance of this cluster formation will be the subject of our further study.

Conclusion
In this paper new electrochemical procedures have been elaborated for the first time to investigate the stability of ascorbic acid (H 2 Asc). It has been proven that the potentiometric titration (PT) experiments supported by the theoretical (modeling) calculations can be successfully applied in a regular analytical practice as well as in the chemical and pharmaceutical industries to control the quality of the aqueous solutions of H 2 Asc. In addition, the elaborated paraffin-protection-layer procedure has been proposed as a good laboratory practice for protecting the solutions against external factors exemplified by: O 2 , CO 2 and temperature (evaporation). Furthermore, the potentiometric titration (PT) method enables us to get some insight into the degradation products of the investigated acid. It has been found that 2,3-diketogulonic acid (2,3-DKGA) is the main product of dehydroascorbic acid (DHA) hydrolysis. Experimental data and the numerical EQSOL procedure confirmed for the first time that 2,3-DKGA exists in an aqueous solution mainly in an enol form with the values of dissociation constants equal pK a3 ~ 3.0 (-COOH) and pK a4 ~9.0 (the -OH enol). Finally, it has been well documented that in aqueous solutions of different electrolytes (KNO 3 , KClO 4 and KSCN) SCN − ions are most effective to protect H 2 Asc against degradation (oxidation). The stabilizing effect of SCN − has subsequently been confirmed by conductometric measurements (CM) and ESI/MS data.

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