Effects of Formulation Variables and Storage Conditions on Light Protected Vitamin B^sub 12^ Mixed Parenteral Formulations

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Article info

Article History :

Received: 15 March 2014

Revised: 21 April 2014

Accepted: 21 April 2014

ePublished: 10 August 2014

Keywords:

* Parenteral

* Stability

* bicompatibility

* Light protected

* Degradation Kinetics

Abstract

Purpose: In this research the effect of vitamin IL and B6 on cyanocobalamin stability in commercial light protected parenteral fonnulations and upon adding stabilizing agents will be investigated and best formulation composition and proper storage condition will be introduced.

Methods: In this research some additives such as co solvents and tonicity adjusters, surfactants, antioxidants and chelating agents as well as buffer solutions, were used to improve the stability of the parenteral mixed fonnulations of B12 in the presence of other B vitamins (B! and B6). Screening tests and accelerated stability tests were perfonned according to ICH guidelines Q1A (R2).

Results: Shelf life evaluation revealed the best formulation and the proper storage condition. The results indicated the first kinetic models for all tested fonnulations and the optimum pH value was detennined to be 5.8. There was no evidence of B12 loss when mixed with B! and Bó in a medical syringe at room temperature for maximum of 8 hours.

Conclusion: It is necessary to formulate vitamin B12 mixed parenteral solutions using proper phosphate buffers (pH=5.8) and to indicate "Store in refrigerator" on the mixed parenteral fonnulations of vitamin B12 with other B vitamins, which has not been expressed on the label of tested Brand fonnulations at the time of this study.

Introduction

It is clearly proved that vitamin B22 or cyanocobalamin is essential for the healthy survival of human cells and because it is not synthesized in mammalian cells, it can only be obtained from external sources.1

In general, many physical and chemical factors can have a negative effect on stability of vitamins. Water-soluble vitamins are prone to degradation in solutions, particularly when exposed to light. B Vitamins are sensitive to factors such as: light, heat, moisture, oxidizing and reducing agents, acids and or bases. There is plenty of literatme concerning the poor stability of vitamin B22 and it has been reported that the optimum pH for stability of this vitamin is 4-6.5 pH value.2"13 It is well known that aqueous solutions of cyanocobalamin are photoliable and some B and C vitamins accelerate the photodegradation.14

Recently the effect of vitamin B2 on the photolysis of cyanocobalamin in aqueous solutions has been investigated by Alnned et al.15 According to kinetic results they concluded that, vitamin B2 acts as a sensitizer in the photolysis of vitamin B12, and thus causes vitamin B12 instability.15 In another study aqueous cyanocobalamin solutions have been photolysed in the presence of individual B (thiamine HC1, riboflavin. nicotinamide and pyridoxine HCl) and C (ascorbic acid) vitamins under high pressure mercury vapor fluorescent lamp. The TLC results indicated that cyanocobalamin changes to hydroxocobalamine, riboflavin degrades to 4- methy 1-5-(ßhydroxy ethyl) thiazole and 2-methyl-4- amino-5-hydroxymethyl-pyrimidine, riboflavin substituted partially with fonnylmethylflavin, lumichrome, lmmflavin and carboxymethylflavin. Ascorbic acid oxidized to dehydroascorbic acid and Nicotinamide and pyridoxine HC1 did not degrade at all. They concluded that all these reactions are pH dependent and a pH value around 4 is optimum for photostability of all B and C vitamins in pharmaceutical preparations.15

Although poor stability of this vitamin has been previously reported, certain parenteral fonnulations are coimnercialized combining this vitamin with other vitamins especially thiamine (B,) and pyridoxine (B6). Authors detected that some manufacturers use high and illegal overages in their light protected fonnulations to compensate vitamin B12 instability issues. These finding along with the interest in vitamins combination triggered a study of the incompatibility of cyanocobalamin in light protected fonnulations to prove the overall effect of vitamin B, and B6 on cyanocobalamin stability in commercialized parenteral concentrations and also to study the effect of some stabilizing agents in order to designate proper fonnulations and storage conditions.

In this Study, the stability of commercial fonnulations was first investigated and then parenteral fonnulations were designed and prepared using some stabilizing additives such as: chelating agents, cosolvents or tonicity-adjusting agents, antioxidants and different buffer solutions. Proper storage conditions according to ICH guidelines were also evaluated and finally aqueous state degradation kinetics was studied and the predominant model was proposed. In addition possible interaction of vitamin BJ2 in commercial injections with other B complex fonnulations when mixed in a single syringe for subsequent human injection has also been investigated.

Materials and Methods

Materials

Cyanocobalamin (5, 6-Dimethylbenzimidazolyl cyanocobamide). Thiamine (3-[(4-Amino-2-methyl-5- pyrimidinyl) methyl] -5-(2-hydroxyethyl)-4- methylthiazolium chloride) and pyridoxine (5-Hydro xy- 6-methyl-3,4-pyridinedimethanol) were obtained from Sigma-Aldrich, Gennany (Figure 1). Hydroxocobalamine (Sigma-Aldrich, Gennany), Ethylenediaminetetraacetic acid (EDTA), propylene glycol (PG), polyethylenglycole (PEG 400), sorbitan mono oleate and buffering agents (Citrate, Acetate and Phosphate) were provided by Merck- Schuchardt, Hohenbrunn, Gennany. All other chemicals were of HPLC or analytical grade and obtained from Merck- Schuchardt, Hohenbrunn, Gennany. Commercial domestic and foreign parenteral fonnulations containing cyanocobalamin, named Brand-1-3 were acquired from local market.

Formulation Methods

Preparation of Parenteral Formulations for Screening and Stability Test

Composition of each prepared fonnulation lias been shown in Table 1. Additives used were solubilizing or tonicity-adjusting agents (lecithin, polyethylene glycol 400, propylene glycol), a surfactant (S.M.O= Sorbitan mono oleate), an antioxidant (S.F.S= Sodium fonnaldehyde sulfoxylate) and a chelating agent (EDTA). The effect of buffer solutions such as acetate, citrate and phosphate, with different pH values and different concentrations were also studied. It is well known that elevated temperatures are fundamental for screening tests. Based on initial results thermal acceleration of degradation reactions were achieved at 55°C and thus screening tests were perfonned at this temperature for 5 days. Prepared fonnulations (n=3) were degassed with bubbling N2 gas and were filled in light protected ampoules and placed in preheated ovens. The amount of remaining vitamin Bi2 was detennined at the end of each day using UV spectrophotometer. Selected fonnulations were subjected to accelerated stability test according to ICH guidelines.16 Briefly samples (n=3) were stored at 40 and 25°C for 6 months and the remaining vitamin Bi2 and also vitamin Bi and B6 were measured and recorded using a proper HPLC method. Each stability evaluation has been perfonned at least 3 times.

Shelf Life Determination

Instead of the well-known Anhenius Equation, a recent ICH guideline for shelf life detennination introduces a new real time data method with no extrapolation.17 Briefly, the upper and lower accepted USP assay limits for cyanocobalamin injection (115% and 95%) were defined16 and the amount of remaining drag in stability samples (n=3) stored at 25°C, in predetennined time intervals were plotted against time. The results of this test, which was conducted as an accelerated stability test for a product intended for use in refrigerator, were assumed as a 6 month real time data for long tenu study at room temperature. Data were plotted in excel software and the intersect position of the 95% lower one sided confidence interval of mean with the lower accepted assay limit in the monograph (95%) was detected and shelf life was calculated at room temperature.

Commercial Formulations

Accelerated stability test at 40 and 25°C for 6 months was perfonned for 3 commercial fonnulations named Brand-1-3.

Brand 1 and 2 were mixed parenteral fonnulation containing B12, B, and B6 and Brand 3 was a mixed B complex fonnulation containing B,. B2. B6, nicotinamide and dexpanthenol without any cyanocobalamin. The amount of each vitamin (Bi2, Bi and B6) was analyzed at the end of the first week and then each month during the stability test, using proper HPLC methods.

Stability of Mixed Commercial Formulation

In this section the stability of coimnercial pure vitamin B12 injection when mixed in a medical grade syringe with vitamin B complex coimnercial injection (without any vitaminB12 according to the label claim), was studied for 8 hours in 37°C under nonnal laboratory light.

Liquid-State Kinetics Study

Six month stability data for selected prepared fonnulations and also brands were fitted to solution kinetics models (zero. First, Second and Third Order) using excel software.18 Mean absolute percentage enor19 and R-squared were used to select the best degradation kinetics model.

Analytical Methods

UV Detection

UV analysis was perfonned on a Shimadzu 1600 UV- visible spectrophotometer (Shimadzu-1600, Japan).

Calibration was perfonned using standard concentrations of vitamin Bi2 (20-160 pg/ml) in the presence of constant concentrations of vitamin Bi and B6 (each 0.02 mg/ml). Three samples of each formulation were collected on predetermined time intervals, dilution was perfonned using 600pi of the ampoule content (except for fonnulations F2 and F3), into which 2400pl of distilled water was added. Then the UV absorption of prepared solutions against water was detected at 550mn to measure the vitamin B12 quantity. Dilution pattern for fonnulations F2 and F3 which contain vitamin B, and vitamin B6, respectively, was as follows. 150pl of each fonnulation were poured into 3 volumetric flasks and brought to a volume of 250ml using freshly distilled water. Then the UV absorption of diluted solutions was detected at 213mn and 291mn for the detennination of vitamin B, and vitamin B6, respectively. Calibration curve was prepared using different standard concentrations of vitamin B, and B6 in the presence of constant concentrations of vitamin B12

HPLC

A liquid chromatographic system (Knauer, Gennany) comprising of Knauer Kl000 solvent delivery module equipped with a Rheodyne (Cotati, CA) injector and a variable wavelength ultraviolet spectrophotometric detector (Knauer smartline 2500) (PDA) . EZ Chrom Elite version 2.1.7 was used for data acquisition, data reporting and analysis.

Vitamin B, and B6 quantification was perfonned isocratically on the reversed phase VP-ODS column (5 pm, 25 cm><4.6 mm; Shimadzu, Japan). The mobile pitase consisted of methanol-phosphate buffer (10:90) and 0.018M triethylamine, which was adjusted to pH 3.55 with 85% orthophosphoric acid. The mobile phase was vacumn degassed prior to its use. A volume of 1 ml/min was used as the flow rate, and detection was perfonned at 283 mn.2" Calibration range used for vitamin B, mixed with vitamin B6 solutions was 0.0366- 0.165 mg/ml.

Vitamin B12 was determined and measured using a newly introduced isocratic reverse phase method at 360 mn. Samples were injected onto a same column, maintained at ambient temperature. Mobile phase was a mixture of acetonitrile:Phosphate buffer (1:6 v/v) and pH was adjusted to 2.6, using orthophosphoric acid. Flow rate was 1.2 nil/niin. Calibration was performed using standard solutions of vitamin B12 prepared in mobile pliase at concentrations of 0.0103-0.33 mg/ml. Both methods were also applied for Hydroxocobalamine determination.

HPLC method validation

Although there was several methods claiming the simultaneous determination of vitamins Bl, B6 and B12, but none of them uses a simple UV detector or coimnon columns.211"22 Method described by Lebiedzinska et al. introduced UV-visible detection only for the determination of thiamine and pyridoxine but not for cyanocobalamin.2"'22 The other method introduced by Markopoulou et al., was not sensitive enough to quantitate small concentrations used in coimnercial parenteral formulations.21 So a new method using C18 column coupled with UV detection as a conventional reversed phase chromatography was introduced for determination of Bi2 which was sensitive enough to detect small quantities in parenteral formulations. It is a novel and efficient method for determination of vitamin B12 in the presence of other B vitamins using reveres pliase HPLC coupled with UV detection. All validation steps including; chromatography method specificity, linearity, accuracy and precision, limit of detection (LOD) and limit of quantification (LOQ), performed for this method and are described in results section.

Results

Formulation Methods

Screening and Stability Test

Based on HPLC results, the cyanocobalamin content of F15 completely decomposed after 2 days. F9 was excluded from the study because of its lecithin content agglomeration after 24 hours. The percentage of remaining vitamin B12 after 5 days of the screening test is shown in Table 2.

Based on the screening test results 5 formulations (F13A, F14Ai F14A2, F14Ci and F14C2) were selected for performing a 6-month stability test (Table 1). Formulations were assayed after passing the screening test and selection was performed empirically based on the total decomposition amount of each formulation to less than 40 percent of their initial content. The results of a 6-month stability test for selected formulations in 40°C oven (accelerated stability test for a product intended for storage at room temperature), and room temperature (25°C) (accelerated stability test for a product intended for storage in a refrigerator) are shown in Figure 1 and Figure 2, respectively. These charts demonstrate the percentage of remaining vitamin Bi2 calculated using a HPLC method. The test ended when more than 90% loss in the amount of vitamin B12 was detectable.

The percentage of remaining vitamin Bi in selected formulations placed in 40°C oven analyzed using HPLC and is shown in Figure 3. No significant change (more titan 5% loss) was detected in the amount of vitamin B6 in the formulations kept at 40°C oven. The amount of vitamin B, and B6 was also constant during incubation at room temperature.

Shelf Life Determination

Calculated shelf lives of five formulations at room temperature are shown in Table 3.

Commercial Formulations

As it mentioned before the main point in studying the stability of mixed coimnercial formulations was triggered after analyzing the initial content of vitamin B12 in new coimnercial products according to their production date. The majority of original innovators liad used more than 5 percent of labeled claim as allowable overage excess content in their formulations. This may be due to compensation of the subsequent loss during shelf life period. The initial percentage of vitamin Bi2, Bi and B6 in coimnercial formulations according to their label claim in the time of testing (assay date) are listed in Table 4.

The results of the stability test for coimnercial formulations incubated in a 40°C oven are shown in Figure 4. Data for vitamin B, and B6 is not sown as no detectable change (more than 5% loss) was observed in the test samples.

Stability of Mixed Commercial Formulation

The percentage of remaining vitamin Bi2 in light protected mixed coimnercial formulations after 8 hours at 37°C reduced to 97%.

Liquid-State Kinetics Study

The best model for vitamin B12 degradation kinetics in selected formulations kept at 25°C and commercials at 40°C were determined according to mean absolute percentage error and R-squared, using excel software. The results indicated the first order kinetics models for all tested fonnulations.

HPLC Methods

Figure 5 depicts the HPLC chromatogram of Hydroxocobalamin standard solution, mixed standard solution of Bi and B6 and stability Sample (F14C2) detected with the HPLC method.

HPLC Method Validation for Vitamin BI2

The retention time of vitamin B12 was about 6.4 min. Based on HPLC chromatogram the specified B12 peak in stability samples and pure solutions were collected from several HPLC injections and then scanned using UV- spectrophotometer and the purity in stability samples was verified through peak purity analysis using PDA.23

The calibration curve for vitamin Bi2 was prepared using five points in the range of 0.0103-0.33 mg/ml, and the regression parameter was equal to l.For determination of the accuracy and precision of the method four samples with concentrations in calibration range were used. Then the mean observed concentrations, standard deviations, C.V. and accuracy were calculated. Results for intra- assay precision (repeatability) and inter-assay precision are shown in Tables 5 and 6.

LOD and LOQ are two parameters that are related to the sensitivity of HPLC method. The signal-to-noise ratio of 3:1 for LOD and 10:1 for LOQ were taken. The peaks were clearly identifiable and had acceptable precision. In this method the LOQ value for vitamin B12 was 0.009 mg/ml and the LOD was 0.003 mg/ml.

Discussion

Screening and Stability Test

Vitamin B, 2 or 5,6-Dimethylbenzimidazolyl cyanocobamide contains several functional groups which makes it prone to different chemical reactions. These groups can be divided into 4 major parts (Figure 6):

1- Core Ring which contains side chains

2- Beta legend. Adenosine and methyl cobalamine functional groups

3- Nucleotide moiety which is a Benzimidazole moiety that is an alpha glycosidic bonded benzimidazole and bonds coordinately to cobalt atom in the core ring and is essential for biological activity

4- Aminopropanol residue that links between the side chain of the core ring and the Nucleotide moiety.

The reactions that can be specified for core ring are briefly as; Deamidation of the side chains. Amidation of carboxylic acid side chains, lactam formation by cyclization (producing dehdrovitamin B12), Lactone formation, subistitution (halogénations, nitration) and Isomerization. Reduction of Cobalt inside the ring.The nucleotide moiety may undergo different reactions such as; Hydrolysis of the phosphate bond, chemical attaclunent of nucleotide moiety and amiopropanol and finally introduction of hetrocycles by microorganisms and the photolytic cleavage of Co-C bond at beta legend part.24

It lias previously proved that Pyridoxal phosphate (PLP) is the active form of Vitamin B6 and acts as a cofactor in many reactions of amino acid metabolism inside the living human cells, including transamination, deamination, and decarboxylation. Thiamine pyrophosphate (TPP), the activated form of vitamin BI, is also a coenzyme in the catabolism of sugars and amino acids. Based on vitamin Bi2 susceptibility to different chemical reactions, vitamin Bi or B6 may trigger its degradation through several pathways. Ahmed et al showed that in aqueous solutions degradation of vitamin B12 mainly results in hydroxylation of cobalt (hyroxocobalamin) and also different oxidation byproducts.3 This information support the utilization of stabilizing agents such as chelating agents, cosolvents or tonicity-adjusting agents, antioxidants and different buffer solutions.

Results revealed that, pure cyanocobalamin solution (FI) is almost stable, but pure thiamin hydrochloride (Bi) solution (F2) and pure pyridoxine hydrochloride (B6) solution (F3) have encountered significant degradation (Table 2). It can be concluded that in light protected conditions vitamin B, aqueous solutions are more stable than vitamin B6 aqueous solutions.

Feller and Macek performed a study and concluded that decomposition of vitamin Bi2 only occurs in high temperature in the presence of thiamine decomposition products.25 In accordance to previous studies,7"26 combination of cyanocobalamin and vitamin Bi (F4) leads to a remarkable destruction of vitamin Bi2 compared to combination of cyanocobalamin and vitamin B6 (F5) (Table 2).

The effect of vitamin B, and B6 on cyanocobalamin stability in parenteral formulations have been investigated previously.3 Alnned et al. summarized that, "In multi-ingredient (Bl + B6 + B12) preparations cyanocobalamin is unstable and degrades from 28% to 37% with concomitant formation of hydroxocobalamin (1.7% to 25.5%) and oxidation products amounting to 56.4% ±9.3.3

Crystalline vitamin Bi2 is stable in solutions of thiamine hydrochloride and niacinamide at pH 3.5 to 4.5 during prolonged storage at room temperature. Thiamine hydrochloride and its decomposition products, which possess reducing properties, are known to destabilize cyanocobalamin solutions at elevated temperatures.25 This finding can explain the difference in the amount of degradation in FI compared to F4.

Triple-ingredient solution (F6) was degraded much more than previous mentioned formulations (Fi-F5), so it can be suggested that the decomposition products of vitamin Bi and vitamin B6 together have a significant influence on the instability of vitamin Bi2. In a similar study multi- ingredient preparations (vitamin B12 1 mg/ml, B, and B6 100 mg/ml), that were stored at room temperature (25- 28°C) for a period of 12 months under normal laboratory light conditions, showed remarkable degradation percentage in comparison with single vitamin B12 solution.3 In this research the aim was to investigate the effect of allowable additives27 in parenteral solutions in order to make a compatible vitamin Bi2 triplet parenteral solution with vitamin Bi and B6.

To the best of our knowledge no research is done using PEG, PG, S.M.O, S.F.S, and acetate, citrate and phosphate buffers as stabilizing additives in vitamin Bi2 mixed parenteral solutions. Based on results, F15, F13B, F8, F12b, Fll, FIO, F7, F12A, F14B2 and F14B1, decomposed to less than 40 percent of their vitamin B12 content (Table 2).

As the amount of the remaining drag in F6 without any additives was about 40 percent (Table 2), these findings showed that the presence of additives in those has destabilized vitamin Bi2 and according to Table 1, PEG and PG 50% v/v as solubilizing and tonicity-adjusting agents, EDTA 0.05% w/v as a chelating agent, S.M.O 0.025%v/v as a surfactant and S.F.S 10% w/v as an antioxidant cannot be considered as stabilizing agents in the case of this study. Acetate buffer (pH=4.75), citrate buffer 0.1M (pH=3.1) and phosphate buffer 0.05M (pH=7 or 8) all failed to stabilize vitamin B12 in its parenteral fonnulations prepared and tested in this study.

In contrast to completely decomposition of F15 in 2 days, some studies have proven the positive effect controversy arises from comes from other investigations revealing vitamin Bi2 instability in combination with vitamin C as a potent antioxidant agent.8

Others showed the effect of pH in the stability of cyanocobalamin and ascorbic acid, alone or in combination.6 In some mixed vitamins and minerals fonnulations, EDTA is added as a chelating agent that reduces vitamin degradation28 but our results showed otherwise.29

It is obvious that all selected fonnulations contained buffer solutions as their stabilizing additives. Selected fonnulations, incubated at 40°C oven, had decomposed over 80% (Figure 1), and the remaining percentage of vitamin BJ2 after 2 months in F13A, F14A1, F14A2, F14C1 and F14C2was about 0, 4.2, 4.05, 11.3 and 11.2% respectively These findings reject the room temperature as optimum storage condition.

According to ICH guidelines accelerated stability test for a product intended for storage in refrigerator needs a study at 25°C. Fonnulations kept at room temperature (25°C) were evaluated weekly for the first month and monthly for a 6 month period. At the end of the study, the percentage of remaining vitamin B12 for these fonnulations was as below: F13A (60.5%), F14A2 (73.4%), F14Ci (78.6%), F14A1 (80.5%), F14C2 (85.5%) (Figure 5).

F13a containing citrate buffer 0.05M, pH=3.1 liad the most decomposition percentage compared with other selected formulations (about 40%) (Table 1). Among these fonnulations, F14C2 was the most stable fonnulation with the remaining B12 content of about 85.5%.

The amount of vitamin B, and vitamin B6 were also detennined weekly for the first month and monthly for a 6 month period. At the end of 3th month, for fonnulations placed in oven 40°C the amount of vitamin B6 liad no significant decline (more than 5% loss), but surprisingly vitamin Bi content of the fonnulations showed a remarkable reduction.

According to Figure 3 the remaining percentages of vitamin Bi in different fonnulations were as follow; F14A2 (44.8), F14Ci and F14C2 (56%), F14A1 and F13A (61%). It can be concluded that about half of vitamin B, content in fonnulations is destroyed, and it could be an evidence to relate cyanocobalamin decomposition with thiamin hydrochloride.7

Fonnulations evaluated in room temperature liad no significant change (more than 5% loss) in their vitamin Bi and vitamin B6 content, and about 95% of their content was remained intact after a 6-month period.

It is thought that a small percentage of cyanocobalamin degrades to hydroxocobalamin during storage.3 Based on this presumption it was decided to check the presence of hydroxocobalamin that was probably produced during stability test. In this Study, pure hydroxocobalamin solution was prepared and the substance was detected using cyanocobalamin specific HPLC method. A sharp peak appeared in about 2 minutes. Stability sample analysis indicated that hydroxocobalamin may have been present in this sample but unfortunately it was not possible to state this precisely, because the hydroxocobalamin peak in the stability samples containing all three vitamins (B12, Bl and B6) lias been overlapped with B1 and B6 peaks.

Shelf Life Determination

USP specifies a pH value of 4.5-7 for the cyanocobalamin injections17 this is a wide range and does not determine the optimum pH for a mixed vitamin B12 parenteral solution with other B vitamins. Based on results, the more and less stable fonnulations are F14C2 and F13a. It can be concluded that citrate buffer 0.1M (pH=3.1) lias less stabilizing effect compared to phosphate buffer (0.05 M, pH= 5.8). The fact of specifying long expiration date for commercial fonnulations comes from huge overage excess content of their fonnulations. The initial percentage of vitamin Bi2 in coimnercial fonnulations according to their label claim in the time of testing which was during their shelf lives and far from manufacturing date and also expiration dates were detennined as 111.9, 68.2 and 129.5 for Brands 1-3 respectively (Table 4). The B12 content in Brand 3 after 9 weeks storage at 40°C decreased about 80% which is comparable to F14Ci and F14C2.

Commercial Formulations

Stability test was also perfonned on several coimnercial fonnulations. Brand 1 a multi-ingredient domestic fonnulation and Brand 2 an innovator multi-ingredient parenteral fonnulation had a drag content of 68% and 130% of their label claim at the first testing date, respectively (Table 4). HPLC data after 9 weeks of storage at 40°C revealed 19 and 50% of the cyanocobalamin label claim for Brands 1 and 2 respectively. Despite the fact that considerable amount of drag liad been added to Brand 3 as excess drag, the amount of vitamin Bi2 decomposition was significantly high. Vitamin Bi and vitamin B6 content of these Brands liad a little variation from their primary value, and they remained almost intact during the test period, which is comparable to data acquired at this time point for our prepared fonnulations. It is interesting that all tested coimnercial fonnulations had also excess amounts of vitamin B, and B6 in their composition (112, 123 and 130% for Brands 2-4 respectively) (Table 4).

Stability of Mixed Commercial Formulation

Based on analyzed data, mixed coimnercial vitamin Bi2 injection with commercial vitamin B complex injection (without any vitamin Bi2 as expressed in label) in a medical grade syringe ready for injection stored at 37°C under normal laboratory light showed no significant loss of Bi2, Bi and B6 (not more than 5% loss). Thus it may be allowable to mix these vitamins before injection.

Liquid-State Kinetics Study

Analysis of the kinetics data indicated the first order reaction in almost all tested formulations.

HPLC Method Validation for Cyanocobalamin Determination In The Presence Of Vitamin If and B6

A new HPLC method was introduced for determination of cyanocobalamin alone and in the presence of vitamins Bi and B6 and possible degradation products. As described before, it is a novel method that lias passed all the method validation steps and can be widely used to analysis vitamin B12 in mixed parenteral dosage forms in pharmaceutical industries.

Conclusion

According to the data obtained from 25°C, F14Ci and especially F14C2 are both acceptable parenteral formulations, and can be stabilized at refrigerator condition (4-8°C). The finding of this research proposes a new stable formulation and proper storage conditions rather than expelling the mixed preparations. Although ICH guidelines rejects the formulations with more than 5% loss during the stability test but adding an allowable overage dose of cyanocobalamin to formulations, will increase the shelf life in an acceptable manner. Based on stability studies performed on prepared and commercial formulations, the optimum pH value for the mixed parenteral formulations of Bi2 was determined to be 5.8. It is necessary to formulate Vitmain Bi2 mixed parenteral solutions using proper phosphate buffers (pH=5.8) and to indicate "Store in refrigerator" on the mixed parenteral formulations of vitamin B, 2 with other B vitamins, which has not been expressed on the label of tested Brand formulations at the time of this study.

More detailed investigations are needed to fully describe the mechanism of incompatibilities involved in cyanocobalamin instability in the presence of B1 and B6 in light protected aqueous solutions.

Acknowledgments

This paper was extracted from Pharm.D thesis no. 3546 submitted to the Faculty of Pharmacy of Tabriz University of Medical Sciences and financially supported by grant no. 91/77 from the Drag Applied Research Center of the same university. The authors are thankful to Zahravi Pharmaceutical Co. Tabriz, Iran for supply of vitamins.

Conflict of Interest

The authors declare no financial or other conflict of interests.

References

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