Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study

Abstract

The degradation kinetics of 5 × 10⁻⁵ M cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) in the presence of ascorbic acid (AH₂) was studied in the pH range of 1.0–8.0. B₁₂ is degraded to B_12b which undergoes oxidation to corrin ring cleavage products. B_12b alone is directly oxidized to the ring cleavage products. B₁₂ and B_12b in degraded solutions were simultaneously assayed by a two-component spectrometric method at 525 and 550 nm without interference from AH₂. Both degrade by first-order kinetics and the values of the rate constants at pH 1.0–8.0 range from 0.08 to 1.05 × 10⁻⁵ s⁻¹ and 0.22–7.62 × 10⁻⁵ s⁻¹, respectively, in the presence of 0.25 × 10⁻³ M AH₂. The t_1/2 values of B₁₂ and B_12b range from 13.7 to 137.5 h and 2.5–87.5 h, respectively. The second-order rate constants for the interaction of AH₂ with B₁₂ and B_12b are 0.05–0.28 × 10⁻² and 1.10–30.08 × 10⁻² M⁻¹ s⁻¹, respectively, indicating a greater effect of AH₂ on B_12b compared to that of B₁₂. The k_obs–pH profiles for both B₁₂ and B_12b show the highest rates of degradation around pH 5. The degradation of B₁₂ and B_12b by AH₂ is affected by the catalytic effect of phosphate ions on the oxidation of AH₂ in the pH range 6.0–8.0.

INTRODUCTION

Cyanocobalamin (vitamin B₁₂) (B₁₂) and hydroxocobalamin (vitamin B_12b) (B_12b) were originally isolated from liver fractions and Streptomyces griseus (1,2). These vitamins are complex organometallic cofactors associated with cobalamin-dependent enzymes that are involved in different catalytic functions in bacteria and humans (3). B₁₂ deficiency produces profound pathological effects on the blood, nervous system, and other organs. The most prominent effect of B₁₂ deficiency is megaloblastic anemia that is caused by the disruption of DNA synthesis (4). B₁₂ [α-(5,6-dimethyl-bezimidazol-1-yl)cobamide cyanide] and B_12b [Coα-[α-(5,6-dimethylbezimidazolyl)]-Coβ-hydroxocobamide] (5) are chemically related (Fig. 1). The CN group attached to Co³⁺ in the tetrapyrrolic corrin macrocyclic ring in B₁₂ is replaced by an OH group in B_12b. B₁₂ undergoes photolysis to B_12b in aqueous solution which is degraded to irreversible oxidation products (6–11). Similar reactions occur on the chemical degradation of B₁₂ and B_12b in the presence of ascorbic acid (AH₂) (12–14) and may be expressed as follows:

1

2

Fig. 1.

Chemical structures of a cyanocobalamin (B₁₂), b hydroxocobalamin (B_12b), c Cob(II)alamin (B_12r), d ascorbic acid (AH₂), and e dehydroascorbic acid (A)

The effect of thiamine (15,16), nicotinamide (17,18), and riboflavin (19–22) on the photolysis of B₁₂ and B_12b has been investigated. Several studies have been conducted on the incompatibility and interaction of B₁₂ and AH₂ that lead to the loss of B₁₂ in aqueous solution (12,13,23–27). B_12b has been found to be less stable than B₁₂ in the presence of AH₂ (28–30) and is destroyed to form unknown oxidation products (9,12,31,32). 3,3-Dimethyl-2,5-dioxopyrrolidine-4-propionamide and 3,3-dimethyl-2,5-dioxopyrrolidine-4-propionic acid have been identified as the oxidation products of B₁₂ (33). The chemical (34–36) and photodegradation (21,37) of B₁₂ and B_12b has been reported to follow first-order kinetics.

The early studies on the stability and degradation of B₁₂ and B_12b are largely qualitative and no systematic work on the kinetics of degradation of these vitamins in the presence of other vitamins has been carried out. One of the reasons for this approach has been the nonavailability of specific methods for the simultaneous assay of B₁₂ and B_12b in degraded solutions (10,18,19). In view of the occurrence of vitamins as mixtures in pharmaceutical preparations, there is a need to reinvestigate their interactions and degradation profiles to achieve greater stability and therapeutic efficacy. B₁₂ and AH₂ both are components of liquid vitamin B-complex with vitamin C (AH₂) and multivitamin preparations, and B₁₂ is liable to degradation in the presence of AH₂ during manufacture, storage, and use. B_12b is an intermediate product in the degradation sequence of B₁₂ and, therefore, a study of its degradation behavior is of fundamental importance in understanding the stability characteristics of B₁₂. It is necessary to ascertain the role of AH₂ in the degradation of B₁₂ and of its degradation product, B_12b, together with that of B_12b alone. AH₂ is oxidized to dehydroascorbic acid (A) (12,14,38) in these reactions. The present work is based on a kinetic study of the comparison of degradation of B₁₂ and B_12b in the presence of AH₂ over a wide range of pH (1.0–8.0), that covers the pH range employed for the formulation of vitamin preparations. The study throws light on the interaction of AH₂ with B₁₂ and B_12b and the extent of formation of oxidation products in these reactions. The magnitude of rate–pH profiles for the degradation of B₁₂ and B_12b provides an indication of the degree of interaction of AH₂ with B₁₂ and B_12b as a function of pH. The mode of degradation of B₁₂ and B_12b in the presence of AH₂ has also been outlined. The information would be of considerable importance to the chemists and pharmacists in designing vitamin formulations with optimum stability of B₁₂ and B_12b. The chemical structures of B₁₂, B_12b, B_12r, AH₂, and A are shown in Fig. 1.

MATERIAL AND METHODS

Materials

B₁₂, B_12b, AH₂, and A were obtained from Sigma-Aldrich. All reagents and solvents were of the purest form available from BDH and Merck. The following buffer systems were used throughout the work:

1. For degradation reactions:

   KCl / HCl (pH 2.0) and citric acid / Na₂HPO₄ (pH 2.5–8.0); the ionic strength was 0.05 M in each case.

2. For assay:

   CH₃COOH/CH₃COONa, 0.2 M (pH 4.0).

Precautions

The degradation, assay, and thin-layer chromatography of B_12b solutions were carried out in a dark chamber under subdued light. The solutions were protected from light during preparation and handling.

Degradation of B₁₂ and B_12b

A 5 × 10⁻⁵ M aqueous solution of B₁₂ or B_12b (100 ml) was prepared at pH 1.0–8.0 using the appropriate buffer, and sufficient amount of AH₂ was added to produce dilutions in the range of 0.25–1.50 × 10⁻³ M (B₁₂) and 0.05–0.25 × 10⁻³ M (B_12b). The solution was placed in a water bath maintained at 25 ± 1°C in the dark chamber to carry out the degradation. The rate of the reaction was followed by spectrometry.

Thin-layer Chromatography

Thin-layer chromatography (TLC) of the solutions of B₁₂ and B_12b, degraded in the presence of AH₂, was performed on 250-μm silica gel GF₂₅₄ plates (Merck) using the following solvent systems:

B₁₂ and B_12b, (a) 1-butanol/acetic acid/0.066 M KH₂PO₄/methanol (36:18:36:10, v/v) (39); and (b) methanol/water (95:5, v/v) (40). The spots were located visually (red color).

AH₂ and A, (c) acetic acid/acetone/methanol/benzene (5:5:20:70, v/v) (41); and (d) ethanol/10% acetic acid (90:10, v/v) (42). The spots were located under UV (254 nm) excitation (AH₂) using a Uvitech lamp (Cambridge, UK), or by spraying with a 3% aqueous phenylhydrazine solution (orange red color) (A).

Spectral Determination

All spectral determinations on B₁₂ and B_12b and the degraded solutions were carried out on a Shimadzu UV-1601 recording spectrophotometer using quartz cells of 10-mm path length.

Spectrometric Assay

The assay of B₁₂ and its degradation product, B_12b, in degraded solutions in the presence of AH₂ was carried out at by a two-component spectrometric method at 525 and 550 nm (pH 4.0, acetate buffer) (10). The degradation products of B_12b formed by the breakdown of the corrin ring may absorb in the UV region and do not interfere with the assay method. The method was validated in the presence of the highest concentration of AH₂ to ensure their accuracy, reproducibility, and specificity under the experimental conditions used.

RESULTS AND DISCUSSION

Degradation Products of B₁₂, B_12b, and AH₂

TLC has been applied to detect the products formed on the degradation of B₁₂ and B_12b in the presence of AH₂. The experimental conditions for the degradation of these vitamins (5 × 10⁻⁵ M) at pH 1.0–8.0 involved AH₂ concentrations of 0.25–1.5 × 10⁻³ M (B₁₂) and 0.05–0.25 × 10⁻³ M (B_12b). TLC of the degraded solutions of B₁₂ (R_f 0.46 and 0.42 in solvent systems (a) and (b), respectively) showed the presence of B_12b only at all pH values (R_f 0.26 and 0.05 in solvent systems (a) and (b), respectively) whereas no degradation product has been detected in B_12b solutions in the pH range studied.

B₁₂ is known to be converted to B_12b by reducing agents including AH₂ (12–14). B_12b is destroyed more rapidly by AH₂ than B₁₂, with a loss of color to indicate the release of cobalt from the molecule (24) and formation of the corrin ring cleavage oxidation products (9,13,32). These products could not be identified by TLC in this study. The oxidation products of B₁₂ have been identified as 3,3-dimethyl-2,5-dioxopyrrolidine-4-propionamide and 3,3-dimethyl-2,5-dioxopyrrolidine-4-propionic acid (33). In the acid medium, the breakdown products of B₁₂ include mixtures of carboxylic acid, γ-lactone and 1-amino-2-propanol (9,13), which do not absorb in the visible region. An R_f 0.22 and 0.73 in solvent systems (c) and (d), respectively, has been detected for A as an oxidation product of AH₂ (R_f 0.30 and 0.49 in (c) and (d), respectively) in the degraded solutions of B₁₂ and B_12b.

Spectral Characteristics and Color Changes of B₁₂ and B_12b in Degraded Solutions

B₁₂ and B_12b exhibit absorption maxima at 278, 361, and 550 nm and 274, 351, and 525 nm, respectively, in water (5,43). AH₂ absorbs at 265 nm at pH 6.4 (44) and does not interfere in the visible region. The absorption spectra of a B_12b solution (pH 5.0) stored in the presence of 1 × 10⁻³ M AH₂ in the dark are shown in Fig. 2. A slight increase in absorbance in the 450–500 nm region after about 90 min gives an indication of the presence of the reduced form of B_12b (Cob(II)alamin, B_12r), absorbing at 474 nm (45). After about 180 min, the spectrum of the colorless solution showed negligible absorption in the visible region as a result of the oxidation of corrin ring (33).

Fig. 2.

Absorption spectra of hydroxocobalamin solution (5 × 10⁻⁵ M) stored in the presence of ascorbic acid (1.5 × 10⁻³ M). a 0 h, red, b 1.5 h, brownish red, c 3 h, colorless

A B₁₂ solution (pH 5.0) stored in the dark in the presence of AH₂ became slightly lighter in color and gradually turned reddish brown. This was followed by a change in color to yellow, and ultimately, the solution became colorless. These changes were more rapid in the degraded solution of B_12b. The color changes involved in the degradation of B₁₂ have been ascribed to the formation of the reduced form of B₁₂ (B_12r) (red to reddish brown), reaction intermediates (brown to yellow) and corrin ring oxidation products (yellow to colorless) (24). In the presence of air B_12r is slowly oxidized to B_12b (32): similar rapid color changes have been observed in B_12b solutions, degraded in the presence of AH₂. These changes indicate that B₁₂ undergoes degradation to B_12r which may further degrade to colorless oxidation products by the cleavage of the corrin ring or be oxidized to B_12b which could also be transformed to the colorless oxidation products in the presence of AH₂ (32).

Assay of B₁₂ and B_12b in Degraded Solutions

The absorption maxima of B₁₂ and B_12b occur at 361 and 550 nm and 351 and 525 nm, respectively, in aqueous solution (43). The British Pharmacopoeia (5) method for the assay of these vitamins is based on the measurement of their absorbance at 361 and 351 nm, respectively. However, due to overlapping of the absorption bands in this region and in the 525 and 550 nm region, the assay of the individual vitamins is not possible. In order to overcome this problem, a two-component spectrometric method was developed and applied to study the kinetics of photolysis of B₁₂ alone (11,19), and also in the presence of nicotinamide (18) and riboflavin (19,20) and the degradation of B₁₂ in parenteral solutions (31). Since B_12b is also a product of photolysis of B₁₂, both B₁₂ and B_12b can be conveniently assayed by this method in B₁₂ solutions degraded in the presence of AH₂. AH₂ absorbs at 265 nm (pH 6.4) (44) and does not interfere in the simultaneous assay of B₁₂ and B_12b by this method. Since the ultimate degradation of B₁₂ and B_12b results in the cleavage of corrin ring to form the oxidation products, which may absorb in the UV region, the set of wavelength 525 and 550 nm in the visible region has been preferred for the two-component assay of B₁₂ and B_12b in degraded solutions. B_12b has directly been assayed at 525 nm to avoid any interference from degradation products. The assay method has been validated in the presence of AH₂ and has a precision of ±2% (Table I). The results of the assay of B₁₂ and B_12b in a typical degradation reaction carried out at pH 5.0 are given in Table II. The increasing loss of molar balance for B₁₂ and B_12b, with time, is due to the formation of the oxidation products (OP) and is an indication of the destructive effect of AH₂ on both B₁₂ and B_12b. The gradual decrease in the OP/B_12b ratios suggests that B_12b may be produced by more than one pathway. This has been discussed under the section on the mode of degradation of B₁₂. Control solution of B₁₂ and B_12b kept in the dark in the absence of AH₂ did not show any degradation during the period of the reactions.

Table I.

Validation Data (n = 5) for Cyanocobalamin (B₁₂) and Hydroxocobalamin (B_12b)

	B12	B12b
Absorption maxima	550	525
Molar absorptivity (M−1 cm−1)	8.70 × 103	8.64 × 103
Linearity range (M × 10−5)	1.0–5.0	1.0–5.0
Correlation coefficient	0.9998	0.9998
Slope	8700	8640
Intercept	−0.0018	−0.0090
SE (±) of slope	0.0031	0.0040
SE of intercept	0.0032	0.0030
SD of intercept	0.0073	0.0068
Recovery range (%)	98.79–101.03	98.96–101.85
Accuracy (%) ± SD	100.03 ± 0.865	100.19 ± 1.121
% RSD	0.865	1.119
LOD (M × 10−5)	0.276	0.260
LOQ (M × 10−5)	0.835	0.787

LOD limit of detection, LOQ limit of quantification

Table II.

Degradation of Cyanocobalamin (B₁₂) in the Presence of Ascorbic acid (AH₂) at pH 5.0

Time (h)	B12 (M × 105)	B12b (M × 105)	B12 + B12b (M × 105)	OPa (M × 105)	OP/B12b
0	5.00	–	5.00	–	–
2	4.68	0.20	4.88	0.12	0.60
4	4.25	0.47	4.72	0.28	0.60
6	3.85	0.73	4.58	0.42	0.58
8	3.54	0.95	4.49	0.51	0.53
10	3.25	1.21	4.46	0.54	0.45
12	2.96	1.47	4.43	0.57	0.39
14	2.70	1.69	4.39	0.61	0.36
16	2.46	1.90	4.36	0.64	0.34
20	2.13	2.19	4.32	0.68	0.31
24	1.77	2.52	4.29	0.71	0.28
28	1.47	2.79	4.46	0.74	0.26
32	1.24	2.98	4.22	0.76	0.25

Experimental conditions were as follows: initial concentration of B₁₂, 5 × 10⁻⁵ M; concentration of AH₂, 1.5 × 10⁻³ M; degradation time, 32 h; temperature, 25 ± 1°C; storage, in dark

^aOxidation products (OP) of B₁₂ = [initial concentration of B₁₂ – (molar concentration of B₁₂ + molar concentration of B_12b at various time intervals)]

Kinetics of Degradation of B₁₂ and B_12b

B₁₂ is known to degrade to B_12b and B_12b to irreversible oxidation products in aqueous solution in the presence of AH₂. Both reactions are affected by AH₂ and the degradation of B_12b alone is much faster than B₁₂ in the presence of AH₂ as discussed in the above sections. These reactions have been studied at pH 1.0–8.0 to assess the degradation behavior of B₁₂ and B_12b. Therefore, the assay data on the degradation of B₁₂ and B_12b were subjected to kinetic treatment, and it was found that these vitamins degrade by an apparent first-order kinetics in the presence of AH₂. This is in agreement with the previous observations that the chemical degradation of B₁₂ and B_12b follows first-order kinetics (34–36). The apparent first-order rate constants (k_obs) for the degradation of B₁₂ and B_12b are given in Table III and IV, respectively. The values of k_obs indicate that the degradation of B₁₂ and B_12b is promoted by AH₂, and the rate increases with an increase in AH₂ concentration throughout the pH range employed. In order to correlate the values of k_obs with AH₂ concentrations, these values were plotted against the respective AH₂ concentrations for the two vitamins and the second-order rate constants (k₂) for the interaction of AH₂ with B₁₂ and B_12b were determined from the slopes of the straight lines (correlation coefficients 0.996–0.999) (Table V). A comparison of the values of k₂ for B₁₂ and B_12b under the reaction conditions employed shows that the rate of interaction of AH₂ with B_12b is about 20 (pH 1.0) to 100 (pH 5.0) times faster than that of B₁₂. Earlier studies have shown that the degradation of B_12b is faster than B₁₂ in the presence of AH₂ (28,46), but no comparison of the kinetic data in these reactions has been made.

Table III.

Apparent First-Order Rate Constants (k _obs) for the Degradation of Cyanocobalamin (B₁₂) in the Presence of Ascorbic acid (AH₂) at pH 1.0–8.0

pH	k obs × 105 [s−1] ± SDa
	0.25b	0.50b	0.75b	1.00b	1.25b	1.50b
1.0	0.08 ± 0.005	0.10 ± 0.006	0.11 ± 0.006	0.12 ± 0.005	0.13 ± 0.007	0.14 ± 0.009
1.5	0.10 ± 0.006	0.12 ± 0.005	0.13 ± 0.007	0.15 ± 0.008	0.16 ± 0.009	0.18 ± 0.008
2.0	0.11 ± 0.006	0.14 ± 0.008	0.18 ± 0.009	0.21 ± 0.009	0.23 ± 0.008	0.26 ± 0.008
2.5	0.30 ± 0.01	0.35 ± 0.02	0.38 ± 0.02	0.41 ± 0.02	0.45 ± 0.03	0.50 ± 0.01
3.0	0.49 ± 0.03	0.53 ± 0.03	0.58 ± 0.04	0.63 ± 0.04	0.67 ± 0.03	0.72 ± 0.03
3.5	0.77 ± 0.04	0.82 ± 0.04	0.88 ± 0.05	0.94 ± 0.04	0.99 ± 0.04	1.05 ± 0.04
4.0	0.99 ± 0.05	1.06 ± 0.05	1.12 ± 0.05	1.19 ± 0.06	1.26 ± 0.07	1.32 ± 0.04
4.5	1.02 ± 0.06	1.08 ± 0.06	1.14 ± 0.06	1.21 ± 0.05	1.28 ± 0.05	1.36 ± 0.07
5.0	1.05 ± 0.05	1.12 ± 0.05	1.18 ± 0.05	1.26 ± 0.06	1.33 ± 0.08	1.40 ± 0.08
5.5	0.95 ± 0.05	1.04 ± 0.05	1.11 ± 0.07	1.19 ± 0.06	1.27 ± 0.05	1.33 ± 0.06
6.0	0.91 ± 0.04	0.97 ± 0.04	1.03 ± 0.05	1.10 ± 0.04	1.17 ± 0.06	1.22 ± 0.05
6.5	0.49 ± 0.02	0.53 ± 0.02	0.56 ± 0.03	0.60 ± 0.03	0.64 ± 0.03	0.68 ± 0.03
7.0	0.29 ± 0.02	0.31 ± 0.02	0.34 ± 0.02	0.37 ± 0.02	0.40 ± 0.02	0.43 ± 0.02
7.5	0.16 ± 0.01	0.18 ± 0.01	0.19 ± 0.01	0.20 ± 0.01	0.21 ± 0.01	0.23 ± 0.01
8.0	0.11 ± 0.01	0.12 ± 0.01	0.13 ± 0.01	0.14 ± 0.01	0.15 ± 0.01	0.16 ± 0.01

Experimental conditions were as follows: initial concentration of B₁₂, 5 × 10⁻⁵ M; concentration of AH₂, 0.25–1.50 × 10⁻³ M; degradation time, 15 h; temperature, 25 ± 1°C; storage, in dark

^a n = 3

^bAH₂ concentration (M × 10³)

Table IV.

Apparent First-Order Rate Constants (k _obs) for the Degradation of Hydroxocobalamin (B_12b) in the Presence of Ascorbic acid (AH₂) at pH 1.0–8.0

pH	k obs × 105 [s−1] ± SDa
	0.05b	0.10b	0.15b	0.20b	0.25b
1.0	0.04 ± 0.002	0.08 ± 0.004	0.13 ± 0.006	0.17 ± 0.008	0.22 ± 0.01
1.5	0.06 ± 0.003	0.15 ± 0.005	0.23 ± 0.010	0.28 ± 0.142	0.34 ± 0.02
2.0	0.11 ± 0.01	0.28 ± 0.01	0.41 ± 0.02	0.50 ± 0.03	0.64 ± 0.03
2.5	0.17 ± 0.01	0.47 ± 0.02	0.68 ± 0.03	0.80 ± 0.03	1.04 ± 0.04
3.0	0.42 ± 0.03	0.89 ± 0.04	1.40 ± 0.06	1.82 ± 0.08	2.30 ± 0.09
3.5	0.85 ± 0.03	1.76 ± 0.09	2.64 ± 0.10	3.55 ± 0.14	4.47 ± 0.20
4.0	1.24 ± 0.05	2.63 ± 0.14	4.05 ± 0.16	5.37 ± 0.25	6.65 ± 0.28
4.5	1.45 ± 0.06	2.82 ± 0.11	4.25 ± 0.18	5.65 ± 0.23	7.04 ± 0.32
5.0	1.55 ± 0.06	3.04 ± 0.12	4.65 ± 0.19	6.02 ± 0.29	7.62 ± 0.36
5.5	1.42 ± 0.06	2.75 ± 0.14	4.15 ± 0.17	5.48 ± 0.25	6.80 ± 0.37
6.0	0.80 ± 0.03	1.69 ± 0.07	2.54 ± 0.10	3.40 ± 0.16	4.20 ± 0.22
6.5	0.65 ± 0.03	1.30 ± 0.05	2.05 ± 0.12	2.72 ± 0.12	3.45 ± 0.14
7.0	0.52 ± 0.03	1.12 ± 0.05	1.65 ± 0.07	2.24 ± 0.09	2.76 ± 0.13
7.5	0.42 ± 0.02	0.77 ± 0.04	1.21 ± 0.05	1.55 ± 0.06	2.02 ± 0.08
8.0	0.35 ± 0.02	0.63 ± 0.03	0.99 ± 0.05	1.28 ± 0.06	1.60 ± 0.08

Experimental conditions were as follows: initial concentration of B_12b, 5 × 10⁻⁵ M; concentration of AH₂, 0.05–0.25 × 10⁻³ M; degradation time, 15 h (pH 1–2), 3 h (pH 2.5), 1 h (pH 3.0–6.5), 3 h (pH 7.0–8.0); temperature, 25 ± 1°C; storage, in dark

^a n = 3

^bAH₂ concentration (M × 10³)

Table V.

Second-Order Rate Constants (k ₂) for the Interaction of Ascorbic acid (AH₂) with Cyanocobalamin (B₁₂) and Hydroxocobalamin (B_12b) at pH 1.0–8.0

pH	k 2 [M−1 s−1] ± SDa
	B12 × 102	B12b × 102
1.0	0.05 ± 0.002	1.10 ± 0.06
1.5	0.06 ± 0.003	1.36 ± 0.05
2.0	0.11 ± 0.004	2.56 ± 0.11
2.5	0.15 ± 0.008	4.20 ± 0.19
3.0	0.19 ± 0.007	9.20 ± 0.51
3.5	0.22 ± 0.009	17.88 ± 0.72
4.0	0.25 ± 0.012	26.60 ± 0.99
4.5	0.27 ± 0.014	29.02 ± 0.99
5.0	0.28 ± 0.013	30.08 ± 1.20
5.5	0.27 ± 0.015	27.20 ± 1.09
6.0	0.24 ± 0.009	16.81 ± 0.67
6.5	0.16 ± 0.008	13.80 ± 0.65
7.0	0.10 ± 0.006	11.04 ± 0.49
7.5	0.06 ± 0.002	8.08 ± 0.34
8.0	0.05 ± 0.002	6.40 ± 0.29

Experimental conditions were as follows: initial concentration of B₁₂ and B_12b, 5 × 10⁻⁵ M; concentration of AH₂, 0.25–1.5 × 10⁻³ M (B₁₂) and 0.05–0.25 × 10⁻³ M (B_12b); degradation time, 1–15 h; temperature, 25 ± 1°C; storage, in dark

^a n = 3

Effect of pH

The pH has a significant effect on the degradation of both B₁₂ and B_12b in the presence of AH_2. The rate–pH profiles give an indication of the interactions between drugs and the rate at which different ionic/non-ionic species undergo degradation as a function of pH. These profiles are helpful in the prediction of the pH of maximum stability of drug substances for the formulation of liquid preparations (38,47). The k₂versus pH profiles for the degradation of B₁₂ and B_12b in the presence of AH₂ are shown in Figs. 3 and 4, respectively. Both profiles are bell-shaped with maxima around pH 5.0. Such profiles imply acid/base dissociation in the reactants. This is possible in the case of AH₂ (pK_a, 4.17) (44) whose monoanion AH⁻ is the reactive species and its concentration would determine the rate of interaction between AH₂ and B₁₂ or B_12b. As the reaction of AH₂ species (AH₂ and AH⁻) with B₁₂ and B_12b proceeds, the formation of AH⁻ anions is increased with pH and hence the rate of interaction between AH₂ and B₁₂ or B_12b, reaching a maximum around pH 5.0. A decrease in pH from 5.0 to 1.0 leads to an increase in the nonionic species of AH₂ and thus the resultant decrease in the rate of interaction. Moreover, the protonated form of B₁₂ (pK_a 3.3) (48) may be less susceptible to degradation than the neutral form. An increase in pH tends to destroy the AH⁻ anions by oxidation (49), resulting in a gradual decrease in the rate of interaction between AH₂ and B₁₂/B_12b. A similar rate–pH profile for the degradation of B_12b in the presence of formate anions (pH_max 5.5) has been reported (50), indicating a similarity of the reaction between ascorbate and formate anions as reductants of B₁₂ and B_12b, respectively.

Fig. 3.

k ₂–pH profile for the degradation of cyanocobalamin (B₁₂) at pH 1–8

Fig. 4.

k ₂–pH profile for the degradation of hydroxocobalamin (B_12b) at pH 1–8

Effect of Buffer

The buffer salts normally used to maintain the pH of pharmaceutical preparations may lead to the degradation of drug substances by acid-base catalysis (38,47). Phosphate salts are commonly used as buffers in the pH range 6.0–8.0 and have been found to catalyze the degradation of drugs including phenytoin (51), codeine (52), ciclosidomine (53), spironolactone (54), gonadorelin (55), riboflavin (56–59), and formylmethylflavin (60). Since the degradation of B₁₂ in this study has also been carried out in phosphate buffer (pH 6.0–8.0), it was considered necessary to evaluate the catalytic effect of phosphate species on the degradation of B₁₂ in 0.02–0.10 M phosphate buffer in the presence of 1.5 × 10⁻³ M AH₂. The graphs of k_obsversus phosphate concentration are linear with a negative slope (R² = 0.997–0.999) and the second-order rate constants (k₂) for the degradation of B₁₂ in the presence of phosphate species at pH 6.0, 7.0, and 8.0 obtained from the slopes of the plots are 1.04, 0.40, and 0.21 × 10⁻⁴ M⁻¹ s⁻¹, respectively. The values of apparent first-order rate constants, k_o, at zero buffer concentration at pH 6.0, 7.0, and 8.0 obtained from y-intercepts of the second-order plots are 1.74, 0.63, and 0.26 × 10⁻⁵ s⁻¹, indicating the inhibitory effect of phosphate species on the reaction. The decrease in the values of k₂, with pH, suggests that the monovalent phosphate species (H₂PO₄⁻) (pK_a 7.21) are more effective than the divalent phosphate species (HPO₄²⁻) in causing the catalytic degradation of AH₂ which would in turn have a lower effect on the degradation of B₁₂.

In the present work, the degradation studies on B₁₂ and B_12b have been carried out in the presence of AH₂ and the values of pH_max obtained are around pH 5.0 (Figs. 3 and 4). Since the phosphate species present in the buffer system (pH 6.0–8.0) are also catalytic to ascorbate monoanion (AH⁻) in the pH range 5.85–7.22 (61), there is a possibility that the buffer species may catalyze the oxidation of AH⁻ anions thereby inhibiting their effect on the degradation of B₁₂ and B_12b. This has in fact been confirmed by conducting the B₁₂ degradation reactions in phosphate buffer. It is evident that the rates of B₁₂ and B_12b degradation increase with a decrease in buffer concentration due to the presence of a relatively greater amount of AH⁻ anions which would interact with these vitamins. Phosphate species tend to destroy AH⁻ anions above pH 5.0, and their rate of oxidation is minimum at pH 5.6 (62) which is slightly above the pH_max for B₁₂ and B_12b. The greater stability and reactivity of AH₂ at pH 5.0 compared to that of pH 7.0 is due to its lower redox potential at pH 5.0 (E° = −0.117 V) than that at pH 7.0 (E° = +0.058 V) (63). The decrease in the rate of degradation of these vitamins above pH 5.0 (Figs. 3 and 4) is probably due to a gradual decrease in the availability of AH⁻ anions for interaction with B₁₂ and B_12b.

Distribution of Degradation Products of B₁₂

The present study has shown that the degradation of B₁₂ in the presence of AH₂ leads to the formation of B_12b and the oxidation products (OP) at pH 1.0–8.0. The product distribution at 50% degradation of B₁₂ along with the t_1/2 values of B₁₂ and B_12b are given in Table VI. A comparison of the molar concentrations of B_12b and OP at various pH values shows that the ratios of OP to B_12b decrease with pH from 1.81 (pH 1.0) to 0.10 (pH 8.0). This could be due to the simultaneous oxidation of AH₂/AH⁻ species with an increase in pH and thus being less effective in causing the oxidation of B_12b. The greater stability of B₁₂ at pH 1.0–2.0 (97–99% protonated form) (pK_a 3.5) (48) indicates that the protonated form of B₁₂ (B₁₂ H⁺) is resistant to degradation. The values of OP at pH 1.0–2.0 may include certain amount of B_12r which would also be more stable to oxidation in this pH range. The relatively high values of OP and the greater stability of B_12b at pH 1.0–2.0 suggest that these may have a contribution from B_12r. The t_1/2 values for B₁₂ and B_12b in the pH range indicate that B₁₂ is more stable to degradation than B_12b as reported in the earlier studies (28,32,46).

Table VI.

Product distribution at 50% degradation (t _1/2) of cyanocobalamin (B₁₂) solutions at pH 1.0–8.0

pH	t 1/2 (h)	B12 (M × 105)	B12b (M × 105)	OPa (M × 105)	OP/B12b
1.0	137.50 (87.50)b	2.50	0.89	1.61	1.81
2.0	74.03 (30.08)b	2.50	1.26	1.24	0.98
3.0	26.74 (8.37)b	2.50	2.05	0.45	0.22
4.0	14.58 (2.89)b	2.50	2.09	0.41	0.19
5.0	13.75 (2.53)b	2.50	2.14	0.36	0.17
6.0	15.78 (4.58)b	2.50	2.20	0.30	0.14
7.0	44.77 (6.97)b	2.50	2.24	0.26	0.12
8.0	120.31 (12.03)b	2.50	2.29	0.22	0.10

Experimental conditions were as follows: initial concentration of B₁₂ and B_12b, 5 × 10⁻⁵ M; concentration of AH₂, 1.50 × 10⁻³ M (B₁₂), 0.025 × 10⁻³ M (B_12b); degradation time, 15 h; temperature, 25 ± 1°C; storage, in dark

^aOxidation products (OP) of B₁₂ = [initial molar concentration of B₁₂ – (molar concentration of B₁₂ + molar concentration of B_12b at 50% degradation)]

^bThe values in parenthesis are t _1/2 of B_12b

Mode of Degradation

It is well known that B₁₂ is incompatible with reducing agents such as AH₂ and is degraded in its presence at room temperature. B_12b is very unstable in the presence of AH₂ and is destroyed by the release of Co atom with subsequent disappearance of the color (12,24,28,29). These changes result in the cleavage of the corrin ring to form the oxidation products (9,12,28). The degradation of B₁₂ by H₂O₂ (64) and HOCl (65) also results in the discoloration of the solutions and the formation of the oxidation products. In the light of the previous studies and on the basis of the present findings, schemes for the mode of degradation of B₁₂ and B₁₂ in the presence of AH₂ are presented.

Degradation of B₁₂

The degradation of B₁₂ by AH₂ in the pH range 1–8 proceeds slowly with AH₂ species (pK_a1, 4.17) (44) up to around pH 3 (3) and then with an enhancement in the rate up to pH 5 due to the participation of the AH⁻ anions (4). This is followed by a decline in the rate of reaction due to the loss of AH⁻ anions. This process leads to the formation of reduced B₁₂ (B_12r) by one electron transfer depending upon the pH and the redox potential of the B₁₂/B_12r system (E° pH 7.0 = −0.04 V) (66). B_12r is directly oxidized to form B_12b by the replacement of CN⁻ by an OH⁻ group (5). Alternatively, it could undergo cleavage of the corrin ring to produce colorless oxidation products (6). The (5) and (6) reactions may occur simultaneously under the conditions employed. B_12r can also disproportionate to B_12s (Co(I)alamin) and B_12b (7) (13). Thus, B_12b could be generated by more than one pathway ((5) and (7)), whereas OP are formed through B_12b only. This would result in a gradual decrease in the OP/B_12b ratios as observed in the present study (Table II). These reactions are presented in Scheme 1:

3

4

5

6

7

Degradation of B_12b

The degradation of B_12b by AH₂/AH⁻ species (8) follows the same pattern as observed for B₁₂, resulting in the formation of the reduced B_12b (B_12br) (9). Similar to the reactions of B₁₂ given in Scheme 1, B_12br may be oxidized to B_12b (10) or may undergo cleavage of the corrin ring to yield the oxidation products (11). The formation of the oxidation products may depend on the pH and the rate of interaction of AH⁻ anions with the B_12b. The degradation reactions of B_12b are presented in Scheme 2:

8

9

10

11

CONCLUSION

Cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) both degrade in the dark by first-order kinetics in the presence of ascorbic acid (AH₂) at pH 1.0–8.0. A comparison of the kinetic data shows that the degradation of B_12b is much faster than that of B₁₂. The maximum rate of degradation of B₁₂ and B_12b occurs at pH 5.0, indicating the highest rate of interaction of AH₂ with B₁₂ and B_12b at that pH. The protonated forms of B₁₂ and B_12b are less susceptible to degradation as evident from the values of the rate constants at pH 1–3. The enhancement in the rate of reaction in the pH range 1–5 is due to gradual ionization of AH₂ to form the ascorbate monoanion (AH⁻) and the reduction in the rate above pH 5 is a result of the loss of AH⁻ anions by oxidation. The degradation of B₁₂ and B_12b involves the reduction of Co³⁺ to Co²⁺ in the corrin ring by AH₂, followed by the oxidation to B_12b and/or the ring cleavage products. These reactions would depend on the pH, concentration of AH₂ and the redox potential of the Co³⁺/Co²⁺ system.