Enhanced Physical Stability of L-Ascorbic Acid in an Ionic Liquid

Abstract

Ionic liquid (IL) technology was used to enhance the stability of L-ascorbic acid (AA). Pyridoxine was selected as the counter cation for anionic AA in IL. After AA was dissolved in water at 40 °C, its ratio decreased to 3.2% after 7 d. In contrast, the IL formulation showed negligible degradation, with almost no loss of AA even after 28 d. These results suggest that the use of IL enhances the stability of AA.

Introduction

L-Ascorbic acid (AA, I in Fig. 1), with a molecular weight of 176.12 and a calculated cLog P of −2.15 using Chem Draw^®, exhibits antioxidant properties, inhibits melanin synthesis, and regulates skin collagen biosynthesis. Due to its lack of side effects, it is widely used in pharmaceuticals, cosmetics, and foods.^1–3) However, the majority of AA is quickly excreted in the urine after its absorption into the systemic circulation, it has low physical stability, and it readily oxidizes in water.^4–10) In the present study, we focused on enhancing the stability of AA without the use of its prodrugs or derivatives.

Fig. 1. Chemical Structures of AA and Pyridoxine

Several derivatives of AA, such as sodium and magnesium salts of AA phosphate, have been developed.^11,12) These hydrophilic forms of AA are stable in formulations and are converted back to AA in the body,¹³⁾ where they exert their effects. They are stable in formulations above pH 7, but may become turbid and emit a foul odor after long-term storage. Additionally, these derivatives, although stable, have insufficient skin permeability.¹³⁾ Another hydrophilic example is AA 2-glucoside, a stable provitamin that is resistant to light and heat and is converted to AA through its metabolism in the intestinal mucosa.¹⁴⁾ However, this conversion process is prolonged, and skin possesses only a limited amount of the enzyme needed for its conversion. AA palmitate, a lipophilic form of AA, was synthesized to improve skin permeability.¹⁵⁾ However, due to its low stability, AA tetrahexyldecylate was subsequently developed.¹⁶⁾ AA palmitate trisodium phosphate, an amphipathic form of AA, has been shown to exhibit better skin permeability and physical stability.¹⁷⁾

In the present study, we devised an alternative approach to AA stabilization that does not rely on the synthesis of derivatives. Our method involves creating a compound containing AA and assessing its impact on AA stabilization. In other words, we focused on an ionic liquid (IL) consisting of AA and its counter ion pyridoxine (Vitamin B₆, II in Fig. 1) (MW: 169.18, cLog P calculated using Chem Draw^®: −0.77) as a compound with similar stability to AA palmitate trisodium phosphate, and investigated whether it increased the physical stability of AA. Pyridoxine was selected as the counter ion for AA due to its potential to enhance the immune functions of the skin and body when combined with AA. In addition, since it is a small molecule similar to AA, pyridoxine contributes to the high solubility of the resulting IL in various solvents. We previously reported that the skin permeation of IL formed from the anionic substance AA and cationic substance pyridoxine was higher than that of AA alone.¹⁸⁾

ILs are generally defined as salts formed by anionic and cationic substances with a melting point below 100 °C.¹⁹⁾ IL technology has recently been employed to enhance gastrointestinal and transdermal absorption.^20–22) IL of AA and pyridoxine also enhanced transdermal absorption. Moreover, it offers the advantage of easy skin application without the need for solvents. Therefore, the present study examined whether IL technology increases the physical stability of AA.

Experimental

Materials and Methods

AA and 1,3-butanediol (1,3-BG) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Pyridoxine was sourced from Sigma-Aldrich (St. Louis, MO, U.S.A.). Sodium sulfate, employed as a desiccant, was procured from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Special grade ethanol was acquired from Kanto Chemical Co., Inc. (Tokyo, Japan). All other reagents and solvents, of HPLC quality or special grade, were used as received without further purification.

Preparation of Neat IL

The anionic substance AA and cationic substance pyridoxine were mixed at a molar ratio of 1 : 1 and dissolved in ethanol. IL was subsequently obtained by the complete removal of ethanol under reduced pressure using an evaporator. The preparation method and physical properties of IL are described in detail in our previous study.¹⁸⁾

Preparation of Sample Solutions

The stabilities of AA, pyridoxine, and IL were examined in 7 different formulations (Table 1): AA or pyridoxine alone in water (Formulations #1 and 2), AA and pyridoxine (1 : 1) physically mixed in water (#3), neat IL (#4), IL in 1,3-BG (IL conc.: 2 mg/mL, the same as below) (#5), IL in 1,3-BG with sodium sulfate at a concentration of 5% (#6), and IL dissolved in 1,3-BG and water (1 : 1)(#7). We selected 1,3-BG as the solvent for IL due to its lower water content than glycerin and propylene glycol.

Table 1. Remaining Ratio of AA and Pyridoxine in Formulations #1–7 on Day 7 or 28

#	Sample	Tested temp.	Concentration of Day 0 (mmol/L, ±S.D.)		Remaining ratio at Day 7 (%)		Remaining ratio at Day 28 (%)
			AA	Pyridoxine	AA	Pyridoxine	AA	Pyridoxine
1	AA alone in water*	5 °C	0.61 ± 0.005	—	82.4	—	—	—
		25 °C			27.7	—	—	—
		40 °C			3.2	—	—	—
2	Pyridoxine alone in water**	5 °C	—	0.60 ± 0.0005	—	101.9	—	—
		25 °C			—	101.8	—	—
		40 °C			—	101.9	—	—
3	AA + pyridoxine physical mixture (1 : 1) in water*,**	5 °C	0.67 ± 0.001	0.57 ± 0.001	82.9	100.5	—	—
		25 °C			6.5	94.1	—	—
		40 °C			0	89.0	—	—
4	Neat IL***	5 °C	764 ± 18.3	780 ± 16.3	97.5	95.4	100.2	97.3
		25 °C			100.7	94.3	91.0	100.5
		40 °C			103.9	97.1	110.8	107.2
5	IL*** in 1,3-BG without sodium sulfate	5 °C	6.81 ± 0.284	6.63 ± 0.294	95.3	97.6	—	—
		25 °C			81.6	96.8	—	—
		40 °C			57.2	97.6	—	—
6	IL*** in 1,3-BG with sodium sulfate	5 °C	7.72 ± 0.488	8.26 ± 0.039	97.8	101.5	—	—
		25 °C			83.2	102.3	—	—
		40 °C			58.3	101.1	—	—
7	IL*** in 1,3-BG and water (1 : 1)	5 °C	6.59 ± 0.278	6.42 ± 0.266	99.0	104.1	—	—
		25 °C			79.6	102.7	—	—
		40 °C			32.6	100.2	—	—

* The concentration of AA was 100 µg/mL. ** The concentration of pyridoxine was 100 µg/mL. ***IL was prepared at the equimolar amount of AA and pyridoxine.

Stability Experiment

Each formulation was stored at 5, 25, and 40 °C for 7 or 28 d. Formulations #1–5 and 7 were stored in an ampoule tube to prevent moisture evaporation from the formulations and moisture absorption into the atmosphere during storage, whereas Formulation #6 was stored in hermetically sealed vials containing sodium sulfate to ensure moisture absorption into the sample. The concentrations of AA and pyridoxine in each formulation were measured on days 0 (immediately after formulation preparation), 1, 2, 3, 4, and 7. The concentrations of AA and pyridoxine in neat IL were also measured on days 14 and 28.

Assessment Methods of AA and Pyridoxine

AA and pyridoxine concentrations in Formulations #1–3 were directly measured using HPLC, while those in Formulation #4 were assessed using HPLC after dissolution in 10 mL of methanol, followed by dilution with purified water. An HPLC system (Shimadzu Corporation, Kyoto, Japan) and the separation column Inertsil^® NH₂ with a particle size of 5 µm and diameter and length of 4.6 × 250 mm (GL Sciences, Tokyo, Japan) were used. Column and auto injector temperatures were maintained at 40 and 5 °C, respectively. The mobile phase was acetonitrile and purified water (1 : 1) containing 0.1% phosphoric acid, and wavelengths for the detection of AA and pyridoxine were 245 and 290 nm, respectively.

Results and Discussion

Table 1 summarizes the remaining ratio of AA and pyridoxine in Formulations #1–7 stored at 5, 25, and 40 °C on day 7 or 28. The degradation of pyridoxine was not observed under any storage conditions. Figure 2 shows the time course of AA in Formulations #1 and 3 for 7 d. Every profile of AA in the figure showed a first-order reaction rate of degradation. When AA alone was stored in water at 5, 25, and 40 °C for 7 d (Formulation #1, Fig. 2a), the ratios of AA remaining were 82.4, 27.7, and 3.2%, respectively. AA must be stored at a low temperature to retard its degradation rate. When the physical mixture of AA and pyridoxine was stored in water under the same conditions as those described above, the ratios of AA remaining were 82.9, 6.5, and 0%, respectively (Formulation #3, Fig. 2b). A little difference was observed in the Formulations #1 and 3. These differences may be experimental errors. These results suggest that physical mixing with pyridoxine did not strongly affect the stability of AA.

Fig. 2. Degradation Profiles of AA in Formulations #1 and 3

Symbols: ●; data at 5 °C, ▲; data at 25 °C, ■; data at 40 °C. Each data point represents the mean ± standard deviation (S.D.) (n = 3–4). *N.D.: not detected.

The physical stabilities of AA and pyridoxine were assessed in neat IL (Formulation #4) stored for 28 d. Almost no degradation of AA or pyridoxine was observed in the formulation, even at 40 °C (Table 1). The reaction site of AA may have been masked by the IL formation. AA is unstable, whereas 2-O-α-D-glucopyranosyl-L-ascorbic acid, which introduced a glycoside group at the 2-position of AA, and magnesium ascorbyl phosphate, which introduced phosphoric acid, are stable.^23,24) On the other hand, ascorbic acid 6-glycoside, which introduced a glycoside group at the 6-position, is not stable.²³⁾ Pyridoxine is expected to stabilized AA by forming an ion pair with the 2-OH group of AA. In addition to the high physical stability of AA, neat IL is useful because of its easy application to the skin without solvents. The use of IL also allows for the preparation of a highly concentrated AA solution (almost 50% in the present study).

Figure 3 and Table 1 show the stability of AA in Formulations #5–7. No degradation of AA was observed in IL samples (Formulations #4–7) on day 0. The ratios of AA remaining in Formulation #5 stored at 5, 25, and 40 °C on day 7 were 95.3, 81.6, and 57.2%, respectively (Fig. 3a). The marked degradation of AA (57.2%) under storage conditions at 40 °C may have been attributed to trace amounts of oxidation-promoting substances in water absorbed by 1,3-BG. Although we have not quantified the water amount in 1,3-BG, the water content must be low because we used fresh, unopened product with a purity of 98% or higher. The experiment was also conducted using sodium sulfate as a desiccant at a concentration of 5% (Formulation #6, Fig. 3b). However, negligible changes were noted in the stability of AA on day 7 (97.8, 83.2, and 58.3% at 5, 25, and 40 °C, respectively). Schenck et al. reported that the dehydration power of sodium sulfate was less effective in polar solvents.²⁵⁾ On the other hand, the ratio of AA remaining was higher in Formulation #7 than in AA alone in water (Formulation #1, Fig. 2a) at all temperatures tested. The AA stability of IL dissolved in water needs to be compared with the stability of AA alone in water; however, this comparison was difficult because this IL does not dissolve in water. In addition, even if IL is dissolved in water, it easily dissociates to the original AA and pyridoxine. Furthermore, it was challenging to compare IL in 1,3-BG with AA in 1,3-BG because AA was not sufficiently soluble in 1,3-BG to be quantified using HPLC. The present IL technology allows for high AA solubility in 1,3-BG. Since AA is more stable in IL in 1,3-BG (Formulation #5) than in water, this formulation has potential as a future formulation.

Fig. 3. Degradation Profiles of AA in Formulations #5–7

Symbols: ●; data at 5 °C, ▲; data at 25 °C, ■; data at 40 °C. Each data point represents the mean ± S.D. (n = 3–4).

The ratios of AA remaining in Formulation #7 (Fig. 3c) stored at 5, 25, and 40 °C on day 7 were 99.0, 79.6, and 32.6%, respectively. The ratios of AA remaining in Formulation #7 were lower than IL in 1,3-BG alone (Formulation #5). IL starts to dissociate to the original AA and pyridoxine when in contact with water. Since water was present at the concentration of IL in 1,3-BG and water (Formulation #5), AA degradation may be greater. The ratio of AA remaining in Formulation #7 was lower than that in Formulation #5, suggesting that a water-free formulation is suitable for AA formulations.

In conclusion, the present results suggest that IL markedly reduced the degradation of AA. IL formulations of AA and pyridoxine may be directly applied to the skin without water or solvents to increase their skin penetration.¹⁸⁾ In addition, neat IL (Formulation #4) may be applied to the skin without solvents, thereby preventing skin irritation by solvents. Moreover, the present IL comprised approximately 50% AA as well as 50% pyridoxine. Based on the results obtained herein, we concluded that IL increased the physical stability of AA.

Conflict of Interest

The authors declare no conflict of interest.

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