DETERMINATION OF ISOSORBIDE MONONITRATE (ISMN) AND ITS TWO RELATED SUBSTANCES IN ISOSORBIDE MONONITRATE AND SODIUM CHLORIDE INJECTION

DETERMINATION OF ISOSORBIDE MONONITRATE (ISMN) AND ITS TWO RELATED SUBSTANCES IN ISOSORBIDE MONONITRATE AND SODIUM CHLORIDE INJECTION

Liandong Hu ^{1, 2*}, Yanjing Shi ^{1, 2} and Na Gao ^{1, 2}

School of Pharmaceutical Sciences, Hebei University ¹, Baoding, Hebei Province, PR China

Key Laboratory of Pharmaceutical Quality Control of Hebei Province ², Baoding, PR China

ABSTRACT: A high-performance liquid chromatography (HPLC) method has been developed for the determination of isosorbide mononitrate and its related substances in isosorbide mononitrate and sodium chloride injection. An C18 column (5μm, 250 ×4.6 mm) was used for the determination, with methanol and water (25:75,v/v)as the mobile phase at the flow rate of 1 mL min⁻¹. The detection wavelength was 210 nm. The related substances were quantitated versus an external standard. The method was capable of resolving all of the two known related substances. The two related substances were isosorbide dinitrate (ISDN) and 2- isosorbide mononitrate (2-ISMN), respectively. The method was successfully applied to quantify related substances. The limits of detection of isosorbide mononitrate, isosorbide dinitrate and 2-isosorbide mononitrate (2-ISMN) were all 0.3ng. And the limits of quantitation of isosorbide mononitrate, isosorbide dinitrate and 2- isosorbide mononitrate (2-ISMN) were all 0.8ng. The method was found to be accurate, precise, specific, linear and sensitive, for the determination.

HPLC, determination, isosorbide mononitrate, related substances

INTRODUCTION:Nowadays, with the development of science and technology, people’s quality of life improved a lot and people’s life style became colourful. However, disease spectra have significantly changed at the same time. Cardiovascular disease has become the main disease harming human health and quality of life and its morbidity rate presents a rising trend.

Additionally, its disability and mortality rates have increased ¹. In the early 1980s, the company of Biovail first developed the isosorbide mononitrate which is a drug used principally in the treatment of angina pectoris and acts by dilating the blood vessels so as to reduce the blood pressure ².

Isosorbide mononitrate is a nitrate-class drug used for the prophylactic treatment of angina pectoris; that is, it is taken in order to prevent or at least reduce the occurrence of angina ³.

There are several process impurities/related substances associated with the manufacture of Isosorbide mononitrate drug substance. Different process related impurities are observed with various synthetic routes and/or manufacturing processes.

In this paper, we used isosorbide mononitrate and sodium chloride injection as samples for sample-destructive testing, according to the testing standards of isosorbide mononitrate and sodium chloride injection in the Chinese Pharmacopoeia ⁴.

Two of the known isosorbide mononitrate related substances studied here are isosorbide dinitrate and 2- isosorbide mononitrate ⁵. Structures of these related substances and their chemical names are provided in Table 1.

TABLE 1: CHEMICAL NAMES AND STRUCTURES FOR ISOSORBIDE MONONITRATE AND ITS RELATED SUBSTANCES

MATERIAL AND METHODS: HPLC grade methanol was purchased from Kemiou Industrial Co., Ltd, Tianjin, China. Isosorbide mononitrate, isosorbide dinitrate and 2- isosorbide mononitrate reference standards were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. The isosorbide mononitrate and sodium chloride injection were supplied from Shijiangzhuang NBP Pharmaceutical Co. Ltd. 

Chromatography: The HPLC system consisted of a quaternary LC-20AT pump, a SPD-20A UV detector and the chromatographic column was a C₁₈ (5μm, 250×4.6mm). The mobile phase composed of methanol and water (25:75). The detection wavelength for the determination was 210 nm. The injection volume was 20μL. The temperature of the column was room temperature.

Standard solutions ⁶:Isosorbide mononitrate standard solution: Took isosorbide mononitrate reference standards, accurately weighed, dissolved in mobile phase and quantitatively diluted, equivalent to 80μg/mL of isosorbide mononitrate approximately, as the standard solution.

Isosorbide dinitrate and 2-isosorbide mononitrate mixture standard solution: Took isosorbide dinitrate and 2- isosorbide mononitrate reference standards，accurately weighed, dissolved in mobile phase and quantitatively diluted, equivalent to 40μg/mL of isosorbide dinitrate and 2- isosorbide mononitrate respectively，as the standard solution.

Sample preparation: Took isosorbide mononitrate and sodium chloride injection, accurately measured, dissolved in mobile phase and quantitatively diluted, equivalent to 80μg/mL of isosorbide mononitrate approximately.

Placebo: A placebo (blank formulation solution) was prepared consisting of purified water, sodium chloride solution.

System suitability: The system was deemed suitable if the following acceptance criteria were satisfied. The tailing factor for the isosorbide mononitrate peak in the resolution solution was not more than 1.6. The resolution between the two substances peaks was not less than 1.5.

Limit of detection (LOD) and limit of quantitation (LOQ): The LOD and LOQ for analytes were estimated by injecting a series of diluted solutions with known concentrations. Solutions of isosorbide mononitrate and two of its related substances were prepared in duplicate. The signal-to-noise ratios for isosorbide mononitrate and the two related substances were calculated. The LOD was evaluated as the concentration, which produced a peak with a signal-to-noise ratio of about 3. The LOQ was evaluated as the concentration that produced a peak with a signal-to-noise ratio of about 10⁷.

Specificity:

Force degradation studies: Solutions of isosorbide mononitrate formulation were stressed with acidic, basic, oxidative, thermal, and photolytic conditions. Prior to analysis, the acid stressed samples were neutralized with base, and the base stressed samples were neutralized with acid. The force-degraded samples were analyzed using a HPLC system. The conditions of the force degradation studies were listed in Table 2.

TABLE 2: THE CONDITIONS OF THE FORCE DEGRADATION STUDIES

 Accuracy and linearity for isosorbide mononitrate ⁸:

• Recovery: Accuracy of the method was calculated by recovery studies at three levels by standard addition method. Samples of product placebo were spiked with isosorbide mononitrate drug substance at 80, 100, and 120% of the product label claim.
• Linearity: Samples of product placebo were spiked with different concentration isosorbide mononitrate stock solution. The spiked samples were assayed for isosorbide mononitrate content and the determined peak areas were plotted versus the spiked concentrations and a linear regression analysis was performed.

Accuracy and linearity for related substances:

• Recovery: Take isosorbide dinitrate and 2- isosorbide mononitrate standard stock solution，accurately weighed, added to the placebo, dissolved in mobile phase and quantitatively diluted. A composite standard solution containing each of the two related substances was prepared and injected in triplicate.
• Linearity: Solutions of two related substances containing at different concentrations were prepared in duplicate and chromatographed. The signal-to-noise ratios for each component were determined.

Ruggedness: Six replicate samples of the injection solution from the same bulk formulation were prepared and assayed using two different laboratories, analysts, instruments and in different days. The individual assay results for isosorbide mononitrate and for related substances were calculated.

The RSD for the six assays were determined for each laboratory and agreement between the mean results was calculated.

Stability of standard and sample solutions: The stability of isosorbide mononitrate in prepared sample solutions was evaluated for 12h under room condition. The assay values obtained at the end of the storage period were compared to the initial concentrations to evaluate the stability of solutions.

Precision of standard solutions: The standard solutions were injected in triplicate.

RESULTS AND DISCUSSIONS: Various mobile phases and columns were used to arrive at a method that achieved an optimal separation for all the components. The chromatographic method described here separated all the related substances of isosorbide mononitrate.

Limits of detection (LOD) and quantitation (LOQ): Table 4 provides the determined LOD and LOQ values for isosorbide mononitrate and the related substances. The limits of detection of isosorbide mononitrate, isosorbide dinitrate and 2- isosorbide mononitrate were all 0.3ng. And the limits of quantitation of isosorbide mononitrate, isosorbide dinitrate and 2- isosorbide mononitrate were all 0.8ng.

Specificity:

Chromatographic profiles: The specificity of the method was determined by individually chromatographing isosorbide mononitrate and two of its related substances (see Table 3). The chromatograms show that the method was specific and no interferences from the blank. The typical retention times and relative retention times for each component were presented in Table 3. Fig. 1 showed typical chromatograms of isosorbide mononitrate and the two related substances.

 TABLE 3: TYPICAL RETENTION TIME, LOD AND LOQ CONCENTIONS OF COMPONENTS

FIG. 1: CHROMATOGRAPHIC PROFILES OF ISOSORBIDE MONONITRATE AND ITS RELATED SUBSTANCES

Force-degradation studies: The results of the Force-degradation studies were as Table 4.

The application of stress conditions did not generate any degradation products that interfered with the determination of isosorbide mononitrate. The results indicated ndicatedh laboratory and agree-the isosorbide mononitrate peak in all the stressed samples was single pure peak. For the drug product samples that were stressed with thermal condition, no appreciable degradation was observed. However, for the base, acidic and oxidative stressed condition, the unknown degradation peaks observed in the samples.

TABLE 4: FORCED DEGRADATION OF STUDIES OF ISOSORBIDE MONONITRATE

 Accuracy/recovery/linearity for isosorbide mononitrate: The results of the recovery studies show that the method was accurate for the determination of isosorbide mononitrate. The individual isosorbide mononitrate recoveries for placebo samples spiked at 80–120% of label claim ranged from 99.91 to 100.79%.

The overall mean recovery was 100.67%. All recovery results were presented in Table 5. The method was found to be linear for isosorbide mononitrate in the 80-120% of label claim. The correlation coefficient (R²) was 0.9998 and the linear regression equation was presented in Table 8.

TABLE 5: ACCURACY AND RECOVERY FOR ISOSORBIDE MONONITRATE

 Accuracy/recovery/linearity for related substances: The recovery results indicate that the method was also accurate for the determination of the two related substances. The results were shown in table 6 and 7. The relative standard deviation (RSD) for isosorbide dinitrate and 2- isosorbide mononitrate were 0.56% and 0.82% respectively, within the limits. The method was found to be linear with correlation coefficients (R²) of 0.993 and 0.992 for the two related substances, respectively, within the limits. The linear regression data for the two related substances were presented in Table 8.

TABLE 6: ACCURACY AND RECOVERY FOR ISOSORBIDE DINITRATE

TABLE 7: ACCURACY AND RECOVERY FOR 2-ISOSORBIDE MONONITRATE

TABLE 8: LINEAR RANGE, COEFFICIENT OF CORRELATION FOR ISOSORBIDE MONONITRATE AND TWO OF ITS RELATED SUBSTANCES

 Stability of sample solutions: The stability of isosorbide mononitrate and two relative substances in standard solutions was evaluated. Prepared samples and standards had been shown to be stable at ambient conditions for at least 12 hours. No other degradation products were observed for any of the solutions tested (table 9).

TABLE 9: STABILITY OF ISOSORBIDE MONONITRATE AND TWO RELATIVE SUBSTANCES

CONCLUSION: The proposed method was found to be accurate, precise, specific, sensitive and linear for the determination of isosorbide mononitrate and two of its related substances in the injection. The method is therefore suitable for the determination of isosorbide mononitrate and its related substances in isosorbide mononitrate and sodium chloride injection.

ACKNOWLEDGEMENTS: This work was supported by the Talent Introduction Program of Hebei University (No. y2005064), the Medical and Engineering Science Research Center of Hebei University (No. BM201109), Hebei Provincial Natural Science Foundation of China- Shijiazhuang Pharmaceutical Group (CSPC) Foundation (No. H2013201274).

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   How to cite this article:

   Hu L, Shi Y and Gao N: Determination of isosorbide mononitrate (ISMN) and its two related substances in isosorbide mononitrate and sodium chloride injection. Int J Pharm Sci Res 2013; 4(12): 4531-36. doi: 10.13040/IJPSR. 0975-8232.4(12).4531-36

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