TY - JOUR AU - Chokshi, Avani, B AB - Abstract A large number of laboratory studies have reported Nitrite (NO2−) and Nitrate (NO3−) to be among the most common degradation products of the high-explosive Nitroglycerin drug substance. A novel, simple, robust and rapid reversed-phase high-performance liquid chromatography method has been developed for quantification of inorganic Nitrite and Nitrate impurities from Nitroglycerin drug substance. Successful separation was achieved in isocratic elution, using Inertsil C8-3, (250 × 4.6 mm, 5.0 μm) column, with mobile phase consisting of pH 7.0 tetrabutyl ammonium hydrogen sulfate buffer, methanol and acetonitrile (96:02:02, v/v/v). Flow rate was monitored at 2.0 mL min−1 and ultraviolet detection at 220 nm. The present work describes the role of an ion-pair reagent in the separation of polar compounds and liquid–liquid extraction technique for separation of polar and non-polar compounds. Nitroglycerin was subjected to various stress conditions to demonstrate the stability-indicating power of the method. The performance of the method was validated as per present International Council for Harmonisation (ICH) guidelines for specificity, linearity, accuracy, precision, ruggedness and robustness. The developed method can be a valuable alternative to the current ion-exchange chromatographic method mentioned in the literature. To the best of our knowledge, a rapid Liquid Chromatography (LC) method, which separates inorganic Nitrite and Nitrate impurities of Nitroglycerin, disclosed in this investigation was not published elsewhere. Introduction Nitroglycerin, a nitrate ester of glycerol (C3H5(ONO2)3), was originally discovered by Ascanio Sobrero in 1847 and later was employed by Alfred Nobel to produce dynamite in the 1860s. Nitroglycerin is a heavy colorless toxic oil that is so unstable that the slightest jolt or friction can result in spontaneous detonation. Although explosive in the liquid state, the solid state is much less sensitive to shock and therefore more stable (freezes at 13°C). It is obtained by nitrating glycerol and is used in the manufacture of explosives and propellants. Nitroglycerin has been the foremost anti-ischemic agent used in clinical medicine for more than a century. Nitroglycerin is a potent vasodilator of veins, arterial conductance vessels and collaterals that has minimal effects on arteriolar tone. At the cellular level, Nitroglycerin is bio-transformed by a still unknown enzymatic process in endothelial cells, smooth muscle and to some extent platelets, causing it to release the vasodilator and anti-aggregator principle nitric oxide (NO). In various organs and tissues, including blood vessels, Nitroglycerin is extensively metabolized via sequential denitration, yielding dinitrates and mononitrates, NO and inorganic nitrite and nitrate ions (NO2− and NO3−). The formation of NO2− and NO3− was possibly caused by β-hydrogen elimination and/or by substitution of OH− at the secondary C in Nitroglycerin as commonly seen in secondary alkyl nitrates (1, 2). Degradation pathway of Nitroglycerin to Nitrite and Nitrate during hydrolysis is mentioned in Figure 1. Figure 1 Open in new tabDownload slide Degradation pathway of Nitroglycerin. Figure 1 Open in new tabDownload slide Degradation pathway of Nitroglycerin. The listed possible impurities that originate from the route of synthesis and/or chemical decomposition from Nitroglycerin are mentioned in Figure 2. Impurities 1-glycerol mononitrate, 2-glycerol mononitrate, 1,2-glycerol dinitrate and 1,3-glycerol dinitrate are analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) method with a solid phase extraction method with Liquid Chromatography UV Detection (LC-UV) (3). The inorganic nitrate and nitrite impurities are quantified by using an ion-exchange chromatographic method in Nitroglycerin drug substance (4). Most investigators employ either thin layer chromatography–liquid scintillation spectrometry (TLC–LSS) or gas chromatography (GC) with either electron capture detector or mass spectrometry for determination of Nitroglycerin and its degradation products (5, 6). Figure 2 Open in new tabDownload slide Degradation products of Nitroglycerin drug substance. Figure 2 Open in new tabDownload slide Degradation products of Nitroglycerin drug substance. The detection of organic nitrates using liquid chromatography–mass spectrometry (LC–MS) has been reported (7, 8), but the detection of inorganic Nitrite and Nitrate in Nitroglycerin drug substance has not been reported. Although, determination of Nitrite and Nitrate content in water, soil, vegetable, milk and urine samples is reported by using different techniques like spectrophotometric method with diazo coupling method (9), HPLC method with cloud-point extraction (10), ion-exchange chromatography (IC) (11–13), ion-pairing HPLC and ion-trap GC–MS (14) HPLC method with fluorescence detection (15) and capillary electrophoresis (16). Literature survey reveals that there is no RP-HPLC method reported for analysis of inorganic Nitrite and Nitrate impurities in Nitroglycerin drug substance. The developed RP-HPLC method is very rapid and cost-effective and can be used as a valuable alternative method to the current IC method. The downside of IC is that ion-exchange columns typically are much more expensive than reversed-phase columns. Also, because reversed-phase columns have been in use for many years, they are reliable and established, meaning that results will be more reproducible from one column to the next. Based on the facts the study was aimed to develop and validate a simple, economic and rapid analytical RP-HPLC method that can be easily applied in routine analysis for the determination of inorganic Nitrite and Nitrate impurities in Nitroglycerin drug substance. Materials and Methods Chemicals and reagents Nitroglycerin (2% w/w in lactose monohydrate) and related impurities were obtained from Zydus Cadila Healthcare Ltd (Gujarat, India). HPLC grade acetonitrile, methanol, triethylamine and orthophosphoric acid were obtained from Merck (Darmstadt, Germany). Millipore Milli-Q water purification system (Bedford, MA, USA) was used for the preparation of high purity water. Tetrabutyl ammonium hydrogen sulfate salt, sodium nitrite and sodium nitrate were obtained from Merck (Darmstadt, Germany). Chromatographic conditions and equipment Chromatographic separation and quantification of Nitrite and Nitrate impurities were performed using the Agilent HPLC System consisting of a quaternary solvent manager, a sample manager and a diode array detector. The output signal was monitored and processed using Chromoleon Software (Dionex). Separation was achieved on Inertsil C8-3 (250 × 4.6 mm, 5.0 μm) column (GL Sciences). The isocratic LC method employs mobile phase which contains 0.22 M tetrabutyl ammonium hydrogen sulfate buffer (pH 7.0), methanol and acetonitrile (96:02:02, v/v/v). The flow rate of the mobile phase was 2.0 mL min−1. The column temperature was maintained at 25°C, and the detection was monitored at a wavelength of 220 nm. The injection volume was 80 μL. Standard and test solutions were prepared in water, and extraction was performed by using methylene chloride. Sample preparation Standard solution for Nitrite and Nitrate impurities was prepared at a concentration of 1.0 μg mL−1 by dissolving appropriate amount of sodium nitrite and sodium nitrate material in water. Sample solution was prepared at a concentration of 100 μg mL−1 of Nitroglycerin in water, sonicated at 10°C and filtered through Millipore PVDF 0.45 μm syringe filter by discarding the first 3 mL of solution. A total of 10 mL of the standard solution and sample solution was taken into a 15 mL centrifuge tube, respectively, and 3 mL methylene chloride was added and mixed by using a vortexer for 60 s. The above prepared solution was centrifuged at 5000 RPM for 5 min, and upper aqueous layer was used for analysis as shown in Figures 3a–c. Figure 3 Open in new tabDownload slide Typical chromatograms of Nitroglycerin solutions; (a) Blank solution, (b) standard solution (1 ppm of Nitrite and Nitrate) and (c) sample solution of 100 ppm Nitroglycerin. Figure 3 Open in new tabDownload slide Typical chromatograms of Nitroglycerin solutions; (a) Blank solution, (b) standard solution (1 ppm of Nitrite and Nitrate) and (c) sample solution of 100 ppm Nitroglycerin. Method validation The developed analytical method was validated for its acceptable performance to ensure suitability for intended purpose. The validation parameters like accuracy, precision, specificity, detection limit, quantification limit, linearity, range, ruggedness and robustness of experiments were executed. The proposed method was validated as per International Council for Harmonisation (ICH) guidelines (17). Linearity was studied by analyzing the mixed calibration standard solutions at six concentration levels. The linearity solutions were prepared by dissolving appropriate amount of sodium nitrite and sodium nitrate standard in water followed by extraction with methylene chloride to achieve the concentration from limit of quantification (LOQ) to 150% of the specification level. Linear regression equations were plotted with the least squares linear regression method. The limit of detection (LOD) and LOQ values were estimated based on the signal-to-noise ratios of 3:1 and 10:1, respectively. Precision of the method was investigated, taking into consideration its intra- and inter-day precision aspects. Intra-day precision was assessed by carrying out six independent sample solutions on the same day. In the inter-day precision study, six new solutions were made on the different day by a different analyst on a different HPLC system with a different lot of column. Both intra-day precision and inter-day precision were valuated according to the Relative Standard Deviation (RSD) values. Accuracy of the method was measured by spiking pre-analyzed samples with four different concentration levels in triplicate, i.e. at LOQ, 50%, 100% and 150% levels of their specification limit 1.0%. Robustness of the method was verified by introducing small variations in the HPLC parameters, including the flow rate (1.8 and 2.2 mL min−1), column temperature (20°C and 30°C), pH of mobile phase buffer (pH 6.8 and pH 7.2) and percentage of buffer and organic phase (98:01:01, v/v/v and 94:03:03, v/v/v). The capacity of the method to detect the responses of the impurities without interferences was determined employing a diode array detector. Forced degradation study was performed using acid, base, peroxide, thermal, humidity and ultraviolet (UV), which may cause drug degradation to evaluate the specificity of the method. Nitroglycerin drug substance was exposed to acidic degradation and base degradation with 5 mL of 1 N hydrochloric acid solution and 1 N sodium hydroxide solution, respectively, for 8 h at room temperature, followed by neutralization and then diluted to 100 μg mL−1 concentration of Nitroglycerin with water followed by extraction with methylene chloride. The upper aqueous layer was injected to observe the effect of acid and alkali hydrolysis. The samples were exposed to light and UV radiation of 2968040 Lux hours and 103959 watt-hours/square meters in a photolytic chamber. Sample solution for photolytic samples was prepared at concentration of 100 μg mL−1 of Nitroglycerin in water; filtration and extraction were carried out by using methylene chloride. The upper aqueous layer was injected to observe the effect of photolysis. In the oxidative condition, Nitroglycerin drug substance was exposed to 5 mL 3.0% hydrogen peroxide solution at room temperature for 2 h. Sample solution was prepared at concentration of 100 μg mL−1 of Nitroglycerin in water, followed by extraction with methylene chloride and aqueous layer was used for study. For thermal degradation, Nitroglycerin drug substance was heated at 45°C for 10 days. Sample solution for thermal stress study was prepared in water at concentration of 100 μg mL−1, filtered and extracted with methylene chloride, and aqueous layer was used. Results Method development The main target of the chromatographic method is to resolve the degradation products generated during stress studies from the Nitrite and Nitrate peaks. Successful separation was achieved by using mobile phase 0.22 M tetrabutyl ammonium hydrogen sulfate salt buffer (pH 7.0), acetonitrile and methanol in the ratio of 96:02:02 (v/v/v) at a flow rate 2.0 mL min−1, which provides better selectivity and resolution. Inertsil C8-3, 250 × 4.6 mm, 5 μm HPLC column was used as stationary phase for better resolution. The peak shapes and the resolution were very good between Nitrite and Nitrate peaks. Liquid–liquid extraction was introduced in the method to remove interference from Nitroglycerin drug substance. This developed method was validated as per ICH guidelines (17). Method validation Specificity Stress studies of a drug substance can help in identifying likely degradation products, which can, in turn, help in establishing degradation pathways and the intrinsic stability of the molecule. The method can be used to validate the stability-indicating power of the analytical procedures used so that the method can be successfully used as a tool for establishing the shelf life of the product. Nitrate and Nitrite impurities were observed during acid hydrolysis and alkali hydrolysis study. Nitrate impurity increased up to level of 3.0% during peroxide oxidation, while Nitrite observed up to the level of 0.5%. During peroxide oxidation, Nitrite is rapidly oxidized by reaction with hydrogen peroxide as mentioned in the following equation: $${\mathrm{H}}_2{\mathrm{O}}_2+{{\mathrm{NO}}_2}^{-}\to{{\mathrm{NO}}_3}^{-}+{\mathrm{H}}_2\mathrm{O}.$$ Nitrite and Nitrate impurities were also observed during thermal stress and humidity conditions up to level 2.0%. Photo-diode array (PDA) detector (scan range 200–400 nm) was employed to check and ensure the homogeneity and purity of Nitrite and Nitrate peaks in all the stressed sample solutions, and peak purity found passing with more than 990 in Chromeleon software (Dionex). The acceptance criterion for peak purity as per Chromeleon software (Dionex) is “not less than 990”, which means that the purity angle should be less than the purity threshold. The results of forced degradation studies are summarized in Table I. Figures 4a–c illustrate separation observed in the forced degradation study. Table I Results of Specificity/Forced Degradation Study Stress type Stress condition % Of impurity Peak purity Nitrate Nitrite Nitrate Nitrite Acid hydrolysis 1 N Hydrochloric acid solution/room temperature/8 h 0.5% 0.3% 997 996 Alkali hydrolysis 0.1 N Sodium hydroxide solution/room temperature/8 h 1.8% 1.7% 998 997 Peroxide oxidation 3% Hydrogen peroxide solution/room temperature/2 h 2.9% 0.5% 997 998 Thermal degradation 45°C for 10 days 2.1% 1.5% 997 997 Humidity degradation 10 days at 40°C/75%RH 1.9% 1.5% 998 998 Photolytic degradation 2968040 Lux hours, 103959 watt-hours/square meters 0.4% 0.2% 997 997 Stress type Stress condition % Of impurity Peak purity Nitrate Nitrite Nitrate Nitrite Acid hydrolysis 1 N Hydrochloric acid solution/room temperature/8 h 0.5% 0.3% 997 996 Alkali hydrolysis 0.1 N Sodium hydroxide solution/room temperature/8 h 1.8% 1.7% 998 997 Peroxide oxidation 3% Hydrogen peroxide solution/room temperature/2 h 2.9% 0.5% 997 998 Thermal degradation 45°C for 10 days 2.1% 1.5% 997 997 Humidity degradation 10 days at 40°C/75%RH 1.9% 1.5% 998 998 Photolytic degradation 2968040 Lux hours, 103959 watt-hours/square meters 0.4% 0.2% 997 997 Peak purity numbers represented as per Chromeleon software (Dionex). Peak is pure only if the value is more than 990. Open in new tab Table I Results of Specificity/Forced Degradation Study Stress type Stress condition % Of impurity Peak purity Nitrate Nitrite Nitrate Nitrite Acid hydrolysis 1 N Hydrochloric acid solution/room temperature/8 h 0.5% 0.3% 997 996 Alkali hydrolysis 0.1 N Sodium hydroxide solution/room temperature/8 h 1.8% 1.7% 998 997 Peroxide oxidation 3% Hydrogen peroxide solution/room temperature/2 h 2.9% 0.5% 997 998 Thermal degradation 45°C for 10 days 2.1% 1.5% 997 997 Humidity degradation 10 days at 40°C/75%RH 1.9% 1.5% 998 998 Photolytic degradation 2968040 Lux hours, 103959 watt-hours/square meters 0.4% 0.2% 997 997 Stress type Stress condition % Of impurity Peak purity Nitrate Nitrite Nitrate Nitrite Acid hydrolysis 1 N Hydrochloric acid solution/room temperature/8 h 0.5% 0.3% 997 996 Alkali hydrolysis 0.1 N Sodium hydroxide solution/room temperature/8 h 1.8% 1.7% 998 997 Peroxide oxidation 3% Hydrogen peroxide solution/room temperature/2 h 2.9% 0.5% 997 998 Thermal degradation 45°C for 10 days 2.1% 1.5% 997 997 Humidity degradation 10 days at 40°C/75%RH 1.9% 1.5% 998 998 Photolytic degradation 2968040 Lux hours, 103959 watt-hours/square meters 0.4% 0.2% 997 997 Peak purity numbers represented as per Chromeleon software (Dionex). Peak is pure only if the value is more than 990. Open in new tab Figure 4 Open in new tabDownload slide Typical chromatograms of forced degradation study; (a) Acid hydrolysis, (b) alkali hydrolysis and (c) peroxide oxidation; sample concentration, 100 ppm. Figure 4 Open in new tabDownload slide Typical chromatograms of forced degradation study; (a) Acid hydrolysis, (b) alkali hydrolysis and (c) peroxide oxidation; sample concentration, 100 ppm. LOD and LOQ The LOD and LOQ for impurities were determined by measurable response at signal-to-noise ratios of 3:1 and 10:1, respectively, by injecting progressively known concentrations of the standard solutions using the developed HPLC method. The precision study was also carried out at the LOQ level by injecting six individual preparations of impurities and calculating the %RSD of the impurity peak areas. The LOD and LOQ values of Nitrite and Nitrate impurities are mentioned in Table II. Table II Regression and Precision Data Parameter Name of compound Nitrate impurity Nitrite impurity Retention time (min) 9.9 6.2 LOD (μg mL−1) 0.03 0.03 LOQ (μg mL−1) 0.10 0.10 Correlation coefficient 0.9997 0.9998 %RSD of standard injections 0.3 0.2 Intra-day precision (%RSD) 2.4 2.1 Precision at LOQ (%RSD) 3.9 4.1 Tailing factor 1.09 1.06 Theoretical plates 7617 7590 Resolution between Nitrate and Nitrite impurities 8.5 Intermediate precision data %RSD of standard injections 0.3 0.3 Intermediate precision (%RSD) 2.6 2.4 Tailing factor 1.11 1.09 Theoretical plates 7342 7136 Resolution between Nitrate and Nitrite impurities 8.0 Parameter Name of compound Nitrate impurity Nitrite impurity Retention time (min) 9.9 6.2 LOD (μg mL−1) 0.03 0.03 LOQ (μg mL−1) 0.10 0.10 Correlation coefficient 0.9997 0.9998 %RSD of standard injections 0.3 0.2 Intra-day precision (%RSD) 2.4 2.1 Precision at LOQ (%RSD) 3.9 4.1 Tailing factor 1.09 1.06 Theoretical plates 7617 7590 Resolution between Nitrate and Nitrite impurities 8.5 Intermediate precision data %RSD of standard injections 0.3 0.3 Intermediate precision (%RSD) 2.6 2.4 Tailing factor 1.11 1.09 Theoretical plates 7342 7136 Resolution between Nitrate and Nitrite impurities 8.0 Open in new tab Table II Regression and Precision Data Parameter Name of compound Nitrate impurity Nitrite impurity Retention time (min) 9.9 6.2 LOD (μg mL−1) 0.03 0.03 LOQ (μg mL−1) 0.10 0.10 Correlation coefficient 0.9997 0.9998 %RSD of standard injections 0.3 0.2 Intra-day precision (%RSD) 2.4 2.1 Precision at LOQ (%RSD) 3.9 4.1 Tailing factor 1.09 1.06 Theoretical plates 7617 7590 Resolution between Nitrate and Nitrite impurities 8.5 Intermediate precision data %RSD of standard injections 0.3 0.3 Intermediate precision (%RSD) 2.6 2.4 Tailing factor 1.11 1.09 Theoretical plates 7342 7136 Resolution between Nitrate and Nitrite impurities 8.0 Parameter Name of compound Nitrate impurity Nitrite impurity Retention time (min) 9.9 6.2 LOD (μg mL−1) 0.03 0.03 LOQ (μg mL−1) 0.10 0.10 Correlation coefficient 0.9997 0.9998 %RSD of standard injections 0.3 0.2 Intra-day precision (%RSD) 2.4 2.1 Precision at LOQ (%RSD) 3.9 4.1 Tailing factor 1.09 1.06 Theoretical plates 7617 7590 Resolution between Nitrate and Nitrite impurities 8.5 Intermediate precision data %RSD of standard injections 0.3 0.3 Intermediate precision (%RSD) 2.6 2.4 Tailing factor 1.11 1.09 Theoretical plates 7342 7136 Resolution between Nitrate and Nitrite impurities 8.0 Open in new tab Linearity Linearity was evaluated for Nitrite and Nitrate impurities (specification limit 1.0%) with respect to the concentration of Nitroglycerin (100 μg mL−1) and not less than six different concentration levels ranging from LOQ to 150%. Nitrite was found linear in the range of 0.102–1.530 μg mL−1, and Nitrate was found linear in the range of 0.101–1.510 μg mL−1. The Y-intercept, slope and correlation coefficient were calculated for both impurities from linear regression equation. The cross-validated r2 was also calculated and found to be more than 0.99 as summarized in Table II. Precision Intra-day precision of the related substance method was checked by injecting six individual preparations (n = 6) of Nitroglycerin drug substance sample spiked with 0.2% of Nitrite and Nitrate impurities. The %RSD of peak area for Nitrite and Nitrate was observed 2.1% and 2.4%, respectively, in six individual preparations of spiked sample solution as mentioned in Table II. The %RSD of peak area for both intra-day precision and intermediate precision was found within 5%. To prove the method ruggedness, the precision of test method was performed by a different analyst by using a different column and a different HPLC system (Shimadzu LC-2010, quaternary solvent manager) on a different day by using a different lot of column. The %RSD of intermediate precision was found within 5%; this proves method ruggedness (Table II). Accuracy In order to evaluate accuracy of the method for related substances, determination was checked by spiking pre-analyzed samples with four different concentration levels in triplicate, i.e. at LOQ, 50%, 100% and 150% levels of the specification limit 1.0% as per ICH guidelines. The samples were analyzed by the proposed method (n = 3), and results are compared (Table III). Table III Accuracy Study for Nitrate and Nitrite Impurities at Different Levels Recovery levela % Of Recoveryb Nitrite impurity Nitrate impurity LOQ 86.5 ± 2.5 (2.9) 87.9 ± 2.9 (3.3) 50% 93.4 ± 2.5 (2.6) 94.5 ± 2.4 (2.5) 100% 94.7 ± 2.3 (2.4) 95.1 ± 2.5 (2.6) 150% 95.4 ± 2.0 (2.1) 96.3 ± 2.4 (2.5) Recovery levela % Of Recoveryb Nitrite impurity Nitrate impurity LOQ 86.5 ± 2.5 (2.9) 87.9 ± 2.9 (3.3) 50% 93.4 ± 2.5 (2.6) 94.5 ± 2.4 (2.5) 100% 94.7 ± 2.3 (2.4) 95.1 ± 2.5 (2.6) 150% 95.4 ± 2.0 (2.1) 96.3 ± 2.4 (2.5) aAmount of impurities spiked with respect to 1.0% specification level individually to Nitroglycerin. bMean ± SD (%RSD) for three determinations at each level. Open in new tab Table III Accuracy Study for Nitrate and Nitrite Impurities at Different Levels Recovery levela % Of Recoveryb Nitrite impurity Nitrate impurity LOQ 86.5 ± 2.5 (2.9) 87.9 ± 2.9 (3.3) 50% 93.4 ± 2.5 (2.6) 94.5 ± 2.4 (2.5) 100% 94.7 ± 2.3 (2.4) 95.1 ± 2.5 (2.6) 150% 95.4 ± 2.0 (2.1) 96.3 ± 2.4 (2.5) Recovery levela % Of Recoveryb Nitrite impurity Nitrate impurity LOQ 86.5 ± 2.5 (2.9) 87.9 ± 2.9 (3.3) 50% 93.4 ± 2.5 (2.6) 94.5 ± 2.4 (2.5) 100% 94.7 ± 2.3 (2.4) 95.1 ± 2.5 (2.6) 150% 95.4 ± 2.0 (2.1) 96.3 ± 2.4 (2.5) aAmount of impurities spiked with respect to 1.0% specification level individually to Nitroglycerin. bMean ± SD (%RSD) for three determinations at each level. Open in new tab Robustness To determine the robustness of the developed method, the experimental conditions were purposely altered, and resolution between Nitrite and Nitrate peaks was evaluated. The flow rate of the mobile phase was 2.0 mL min−1. To study the effect of flow rate, it was changed by ±10% from 2.0 to 2.2 and 1.8 mL min−1. Column oven temperature (T) was 25°C, and to study the effect of column oven (T), it was changed by 5.0°C units from 25°C to 30°C and 20°C. The pH of mobile phase buffer was changed (pH 6.8 and pH 7.2), and the percentage of buffer and organic phase was altered (98:01:01, v/v/v and 94:03:03, v/v/v) to observe the system suitability parameters in altered conditions. In all altered condition (flow rate, column temperature, organic composition and pH change), both peaks were well separated and had a resolution more than 5.5 between Nitrite and Nitrate impurity peaks, and there was no change in elution order. Solution stability The solution stability for standard solution and sample solution was examined at different time intervals; the cumulative RSD was calculated for each impurity response to check the consistency. Mobile phase composition and preparation were kept constant during the study period. For standard solution, the peak area of Nitrate and Nitrite impurities were observed at every 6 h of time intervals, and cumulative %RSD with respect to the mean peak area of standard solution after 36 h was found 1.6% for Nitrate impurity and 1.8% for Nitrite impurity. For sample solution, the chromatograms were reviewed to check the response of Nitrate and Nitrite peaks due to the interaction with the analyte in the solution. The cumulative %RSD after 36 h was observed 1.9% for Nitrate impurity and 1.8% for Nitrite impurity and concluded that standard solution and sample solution is stable at room temperature for 36 h. Discussion Optimization of chromatographic conditions The main target of the chromatographic method is to get separation of the degradation products generated during stress studies from the Nitrite and Nitrate peaks. Optimization of chromatographic conditions was carried by using polar stationary phase as Nitrite and Nitrate are very polar in nature. Nitrite and Nitrate impurities were co-eluted by using different stationary phases like cyano and phenyl with different particle sizes and lengths and different mobile phases containing buffers like phosphate and acetate with different pH (2–6) and using organic modifiers like acetonitrile and methanol in the mobile phase. Tetrabutyl ammonium hydrogen sulfate salt was used as an ion-pair reagent and added in the mobile phase to retain the Nitrite and Nitrate impurities with Kromasil C-8, 150 × 4.6 mm with 5 μm particles as stationary phase. The retention of charged solute is a complex function of the nature and the concentration of ion-pair reagent. Stoichiometric theory assumes that the solute ions and the ion-pair reagents form stoichiometric complexes at the surface of the stationary phase, which leads to retention of the compound. The mobile phase contains 0.1 M tetrabutyl ammonium hydrogen sulfate salt buffer (pH 3.0), and flow rate was kept as 1.0 mL min−1. Standard solution was prepared by dissolving appropriate amount of sodium nitrite and sodium nitrate in water, was injected and observed poor resolution between Nitrite and Nitrate peaks with this condition and was not considered for further analysis, as shown in Figure 5a. Later, 0.22 M tetrabutyl ammonium hydrogen sulfate salt buffer (pH 7.0) in water was used as mobile phase, which provides better selectivity and resolution. Column used was Inertsil C8-3, 150 mm × 4.6 mm, 5 μm. Finally, 0.22 M tetrabutyl ammonium hydrogen sulfate salt buffer (pH 7.0) and acetonitrile and methanol in the ratio of 96:02:02 (v/v/v) at a flow rate 2.0 mL min−1 were selected as mobile phase for better stability of mobile phase. Inertsil C8-3, 250 × 4.6 mm, 5 μm HPLC column was used as stationary phase for better resolution. The peak shapes and the resolution were very good between Nitrite and Nitrate peaks. Figure 5 Open in new tabDownload slide Typical chromatograms at 210 nm; (a) Standard solution with 0.1 M ion-pair concentration, (b) organic layer of sample solution and (c) aqueous layer of sample solution. Figure 5 Open in new tabDownload slide Typical chromatograms at 210 nm; (a) Standard solution with 0.1 M ion-pair concentration, (b) organic layer of sample solution and (c) aqueous layer of sample solution. In this chromatographic condition, Nitroglycerin peak elutes very late at about 400 min due to high affinity with the ion-pair reagent. Gradient program was applied by increasing organic phase in the mobile phase B; the equilibration was not achieved in this condition, and variation in the retention time was observed with run-to-run variation. So many attempts were taken to remove Nitroglycerin from the sample solution without affecting the Nitrite and Nitrate content. Finally, methylene chloride was used for extraction of Nitroglycerin from the sample due to high affinity for organic phase, while Nitrite and Nitrate remain in the aqueous phase due to their polar nature. After extraction, lower organic layer was evaporated to dryness, and the residue was dissolved in methanol and injected in the assay method of Nitroglycerin. The response of Nitroglycerin observed in the organic layer suggests that the drug substance was present in the organic phase (Figure 5b). Upper aqueous layer was injected in the same method, and no peak observed at the retention time of Nitroglycerin proves that Nitroglycerin transferred to organic layer from upper the aqueous layer, as shown in Figure 5c. This extraction technique was used for sample preparation, and the developed method was validated as per ICH guidelines (17). Method validation Nitrate and Nitrite impurities are degradation-related impurities of Nitroglycerin, and these impurities are observed during stress study. During stress study, ring cleavage occurs as mentioned in Figure 1. Homogeneity and peak purity for both the peaks were observed by using PDA detector in all the stressed sample solutions, and peak purity was found passing with more than 990. Therefore, it can be concluded that the method is specific. LOD and LOQ LOD is defined as the lowest amount of analyte that can be detected but not necessarily quantified as an exact value, whereas LOQ is defined as the lowest amount of analyte that can be determined with suitable precision and accuracy. Linearity The linearity of an analytical procedure is its ability to obtain test results that are directly proportional to the concentration of the analyte in the sample. The result showed that an excellent correlation existed between the peak area and concentration of the analyte. This confirmed the linear relationship between peak areas and concentrations. Precision System repeatability was determined from six replicate injections of by injecting six individual preparations (n = 6) of Nitroglycerin drug substance sample spiked with 0.2% of Nitrate and Nitrite impurities by two different analysts with different chromatographic systems on two different days. The RSD value of repeatability/intermediate precision shows that the proposed method provides acceptable precision. Accuracy Accuracy of an analytical procedure expresses the closeness of agreement between the values, which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy results are within the acceptance criteria of 80.0% to 120.0%, which suggests that the method is accurate. Robustness The robustness of an analytical procedure provides an indication of its reliability during normal use. In all altered condition (flow rate, column temperature, organic composition and pH change), both peaks were well separated and had a resolution more than 5.5 between Nitrite and Nitrate impurity peaks; there was no change in elution order. The results confirmed that the proposed method is robust. Solution stability The results from the solution stability experiments confirmed that the standard solutions and test solutions in the diluent were stable for up to 36 h at room temperature during the quantification of Nitrate and Nitrite impurities in Nitroglycerin drug substance. Application of the method The validated method was applied for the impurity analysis in three lots of drug substance. Nitrite impurity was not detected in all of the samples, while Nitrate impurity was found in Nitroglycerin drug substances at level of about 0.3%. The proposed method can be successfully applied in the quality control for analysis of Nitroglycerin drug substance as an alternative to ion-exchange chromatographic method. Conclusion A validated RP-HPLC method was developed for quantitative determination of inorganic nitrite and nitrate impurities in Nitroglycerin drug substance. All the degradation products were well separated from the Nitrite and Nitrate peaks as the degradation products are extracted by using methylene chloride, demonstrating the stability-indicating nature of the proposed method. The RP-HPLC method is simple, robust, accurate and selective. The method was completely validated as per ICH guidelines and results from validation confirm that the method can be used for its intended purpose. Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) TI - Liquid Chromatographic Method Development for Quantification of Inorganic Nitrite and Nitrate Impurities from Nitroglycerin Drug Substance by Using Ion-Pair Reagents with Liquid–Liquid Extraction Technique JF - Journal of Chromatographic Science DO - 10.1093/chromsci/bmz102 DA - 2020-01-01 UR - https://www.deepdyve.com/lp/oxford-university-press/liquid-chromatographic-method-development-for-quantification-of-0COGxsrAnN DP - DeepDyve ER -