A sensitive, stability-indicating gradient RP-HPLC method has been developed for the simultaneous estimation of impurities of Guaifenesin and Dextromethorphan in pharmaceutical formulations. Efficient chromatographic separation was achieved on a Sunfire C18, 250 × 4.6 mm, 5 µm column with mobile phase containing a gradient mixture of solvents A and B. The flow rate of the mobile phase was 0.8 mL min−1 with column temperature of 50°C and detection wavelength at 224 nm. Regression analysis showed an
Guaifenesin (GN), (+)-3-(2-methoxyphenoxy)-propane-1,2-diol, is a widely used expectorant, useful for the symptomatic relief of respiratory conditions. Its empirical formula is C10H14O4, which corresponds to a molecular weight of 198.21. It is a white or slightly gray crystalline substance with a slightly bitter aromatic taste. Its solid oral dosage form is available as extended release tablets for oral administration [
Dextromethorphan (DN) [
Structures of Guaifenesin and Dextromethorphan.
Molecule name | Chemical name | Chemical structure |
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Guaifenesin | (+)-3-(2-Methoxyphenoxy)-propane-1,2-diol |
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Dextromethorphan | Ent-3-methoxy-17-methylmorphinan |
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Impurity profiling of active pharmaceutical ingredients (API) in both bulk material and finalized formulations is one of the most challenging tasks for pharmaceutical analytical chemists under industrial environment [
GN and DN are official in the United States pharmacopeia and European pharmacopeia, but its combination is not official in any of the pharmacopeias. In the literature survey, there were several LC assay methods that have been reported for the determination of GN and DN in pharmaceutical preparation either individually or in combination with other drugs [
There is no single method reported for the simultaneous determination of the impurities in pharmaceuticals formulations of GN and DN. It is felt to develop a stability-indicating method for simultaneous determination of GN and DN related impurities in pharmaceutical formulation.
Hence, an attempt has been made to develop an accurate, rapid, specific, and reproducible method for the determination of GN and DN impurities (Tables
(a) Structures of Guaifenesin impurities. (b) Structures of Dextromethorphan impurities.
Impurity name | Chemical name | Chemical structure |
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Impurity A (degradant) | 2-Methoxyphenol |
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Impurity B (degradant) | 2-(2-Methoxyphenoxy)propane-1,3-diol |
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Impurity C (degradant) | 1,1′-Oxybis[3-(2-methoxyphenoxy)propan-2-ol] |
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Impurity D (process related impurity) | 1,3-Bis(2-methoxyphenoxy)propan-2-ol |
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Impurity name | Chemical name | Chemical structure |
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Impurity A (degradant) | Ent-3-methoxymorphinan |
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Impurity B (degradant) | Ent-17-methylmorphinan-3-ol |
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Impurity C (degradant) | Ent-3-methoxy-17-methylmorphinan-10-one |
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N-Oxide (degradant) | 3-Methoxy-N-methylmorphinan N-oxide |
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N-Formyl Morphine (NFM) (process related impurity) | 3-Methoxy-6,7,8,8a,9,10-hexahydro-5H-9,4b-(epiminoethano)phenanthrene-11-carbaldehyde |
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N-Formyl octabase (NFO) (process related impurity) | (S)-1-(4-Methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline-2(1H)-carbaldehyde |
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GN + DN tablets were received from the formulation research and development laboratory of Dr. Reddy’s Laboratories Ltd., IPDO, Hyderabad, India. GN API and impurities were procured from Synthochem Lab., India. DN API and impurities were procured from Wochardt Laboratories Ltd., India. Sodium dihydrogen phosphate monohydrate, 1-octane sulfonic acid sodium salt monohydrate, HPLC grade acetonitrile, methanol, and orthophosphoric acid were purchased from Merck, Germany, Regis Technologies Inc, USA, and highly pure water was prepared by using Millipore MilliQ plus purification system.
The LC system used for method development and method validation was Waters with a diode array detector (model: 2998 detector and e2695 separation module). The output signal was monitored and processed using Waters Empower software. Weighing was performed with a Mettler XS 205 Dual Range (Mettler-Toledo GmbH, Greifensee, Switzerland). Photo stability studies were carried out in a photo stability chamber (SUN TEST XLS+, Atlas, USA). Thermal stability studies were performed in a dry air oven (Merck Pharmatech, Hyderabad, India).
HPLC measurements were carried out using a reversed phase Sunfire, C18, 250 × 4.6 mm, 5
We prepared individual stock solutions for GN, DN, and their impurities (each 500
Twenty tablets (1200 mg of GN + 60 mg of DN) were weighed and the average weight was calculated. The tablets were crushed into fine powder, and powder equivalent to 6000 mg of GN (or equivalent to 300 mg of DN) was transferred into a 250 mL volumetric flask. Approximately 170 mL of diluent was added, shaked to disperse the material, and sonicated for 20 minutes with intermediate shaking. The solution was then diluted to 250 mL and centrifuged at 3000 rpm for 10 min. The supernatant (24000
The proposed method was validated as per ICH guidelines [
System suitability parameters were evaluated to verify the system performance. System precision was determined on six replicate injections of standard preparations. All the important characteristics, including the relative standard deviation, peak tailing, and theoretical plate number, were measured. Resolution between impurities was measured by injecting system suitability solution. All these system suitability parameters covered the system, method, and column performance.
Stress studies were performed at an initial concentration of 24000
The precision of the determination of the impurities was checked by injecting six individual preparations of (24000
LOD and LOQ for all impurities including GN and DN were determined at a signal-to-noise ratio of 3 : 1 and 10 : 1, respectively, by injecting a series of dilute solutions with known concentrations. Precision study was also carried out at LOQ level by injecting six individual preparations of impurities and % RSD was calculated.
Linearity test solutions for the method were prepared by diluting stock solution to the required concentrations. The solutions were prepared at six concentration levels from LOQ to the target test concentration. The peak area versus concentration in
Accuracy of the method was evaluated by using concentration levels LOQ, 0.1%, 0.2%, 0.4%, 0.8%, and 1.0% on GN + DN tablets. Six preparations were performed at LOQ and 1.0% level and three preparations were performed at different levels. Standard addition and recovery experiments were conducted on real sample to determine accuracy of the related substance method. The percentages of recoveries for all impurities, GN, and DN were calculated.
To determine the robustness of the developed method, experimental conditions were deliberately changed and the resolution between GN, DN, and their impurities, tailing factor, and theoretical plates of GN and DN peaks were evaluated. Also relative retention times for all the impurities and column pressure throughout the run were monitored.
To study the effect of the flow rate on the developed method, it was changed from 0.8 mL min−1 to 0.6 and 1.0 mL min−1. The effect of column temperature on the developed method was studied at 45 and 55°C (instead of 50°C). The effect of pH was studied by varying ±0.2 pH units (i.e., 2.8 and 3.2) and the mobile phase composition was changed ±10% from the initial composition. In all the above varied conditions, the component of the mobile phase was held constant.
GN and DN spiked samples (impurities spiked at 0.2% of the target test concentration, i.e., 48
The main criterion was developing an RP-HPLC method for the simultaneous determination of impurities in GN and DN pharmaceutical dosage form in a single run, with emphasis on the method being accurate, reproducible, robust, stability-indicating, linear, free of interference from other formulation excipients, and convenient enough for routine use in quality control laboratories.
Individual stock solutions of GN, DN, and their impurities were injected and the spectra were checked of each component (Figure
Spectra of GN, DN, and their impurities.
A spiked solution of impurities (48
Method development was initiated by changing different gradient programmes, different pH values of the mobile phase buffer, different phosphate buffers, and different columns with the literature method [
The chromatographic separation was achieved by a reversed phase Sunfire, C18, 250 × 4.6 mm, 5
Overlaid chromatogram of blank and system suitability preparation.
Overlaid chromatogram of placebo and spiked test preparation.
After the development of the method it was subject to method validation as per ICH guidelines [
The percentage relative standard deviation (RSD) of area from six replicate injections was below 5.0% (diluted standard solution, 48
System suitability results.
Parameter | % RSD* of standard | Theoretical plates* | Tailing factor* | Resolution 1 | Resolution 2 | |||
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GN | DN | GN | DN | GN | DN | |||
As such method | 0.5 | 0.7 | 45320 | 498659 | 1.0 | 1.0 | 2.7 | 2.3 |
At 0.6 mL/min flow rate | 1.2 | 0.6 | 41091 | 495497 | 1.0 | 1.0 | 2.9 | 2.5 |
At 1.0 mL/min flow rate | 0.9 | 0.4 | 45828 | 515551 | 1.0 | 1.0 | 2.5 | 2.2 |
At 50°C column temperature | 1.5 | 0.8 | 45321 | 508966 | 1.0 | 1.0 | 2.6 | 2.3 |
At 60°C column temperature | 2.1 | 2.3 | 40929 | 515807 | 1.0 | 1.0 | 2.9 | 2.6 |
At pH 2.8 (buffer pH) | 1.1 | 0.5 | 43970 | 463987 | 1.0 | 1.0 | 2.4 | 2.3 |
At pH 3.2 (buffer pH) | 0.7 | 1.5 | 44619 | 532891 | 1.0 | 1.0 | 2.6 | 2.7 |
All forced degradation samples were analyzed with the aforementioned HPLC conditions using a PDA detector to monitor the homogeneity and purity of the GN, DN, and their related impurities. Individual impurities, placebo, GN, and DN were verified and proved to be noninterfering with each other thus proving the specificity of the method.
Figure
(a) Forced degradation data for Guaifenesin. (b) Forced degradation data for Dextromethorphan.
Degradation conditions | Guaifenesin | |||
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% degraded | Purity angle | Purity threshold | Mass balance (%) | |
Photo. stress (1.2 million lux hours followed by 200 Watt hours) | 0.06 | 3.585 | 4.142 | 100.2 |
Exposed to humidity at 25°C and 90% RH for about 7 days | 0.07 | 3.881 | 4.241 | 99.8 |
Refluxed with purified water for about 12 hours at 60°C | 0.05 | 3.285 | 3.845 | 98.6 |
Refluxed with 0.5 N HCL solution for about 2 hours at 60°C | 0.10 | 5.293 | 7.073 | 99.2 |
Refluxed with 0.5 N NaOH solution for about 2 hours at 60°C | 0.06 | 3.067 | 3.489 | 98.9 |
Refluxed with 10% H2O2 solution for about 30 min at 60°C | 0.13 | 2.755 | 3.607 | 98.3 |
Exposed to dry heat for about 15 hours at 105°C | 0.07 | 3.346 | 4.331 | 99.4 |
Degradation conditions | Dextromethorphan | |||
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% degraded | Purity angle | Purity threshold | Mass balance (%) | |
Photo. stress (1.2 Million lux hours followed by 200 Watt hours) | 0.01 | 0.115 | 0.242 | 99.8 |
Exposed to humidity at 25°C and 90% RH for about 7 days | 0.01 | 0.124 | 0.261 | 99.5 |
Refluxed with purified water for about 12 hours at 60°C | 0.01 | 0.101 | 0.261 | 98.9 |
Refluxed with 0.5 N HCL solution for about 2 hours at 60°C | 0.02 | 0.154 | 0.262 | 98.1 |
Refluxed with 0.5 N NaOH solution for about 2 hours at 60°C | 0.01 | 0.103 | 0.256 | 99.2 |
Refluxed with 10% H2O2 solution for about 30 min at 60°C | 1.05 | 0.102 | 0.250 | 99.7 |
Exposed to dry heat for about 15 hours at 105°C | 0.01 | 0.099 | 0.261 | 98.7 |
Typical chromatogram of peroxide stressed sample.
The % RSD for the individual % of all impurities in impurities method precision study was within 3.4%. The results obtained in the intermediated precision study for the % RSD of the individual % of all impurities were well within 4.1%, conforming high precision of the method. The results are shown in Tables
Results of precision.
Compound | Prep-1 | Prep-1 | Prep-3 | Prep-4 | Prep-5 | Prep-6 | Avg | % RSD |
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GN-Impurity A | 0.198 | 0.201 | 0.189 | 0.213 | 0.205 | 0.209 | 0.203 | 4.2 |
GN-Impurity B | 0.209 | 0.195 | 0.192 | 0.201 | 0.206 | 0.194 | 0.200 | 3.5 |
GN-Impurity C | 0.189 | 0.195 | 0.186 | 0.192 | 0.198 | 0.184 | 0.191 | 2.8 |
GN-Impurity D | 0.208 | 0.190 | 0.192 | 0.193 | 0.188 | 0.186 | 0.193 | 4.1 |
DN-Impurity A | 0.215 | 0.214 | 0.215 | 0.209 | 0.212 | 0.205 | 0.212 | 1.9 |
DN-Impurity B | 0.204 | 0.200 | 0.202 | 0.203 | 0.205 | 0.191 | 0.201 | 2.5 |
DN-Impurity C | 0.195 | 0.186 | 0.198 | 0.199 | 0.184 | 0.194 | 0.193 | 3.2 |
DN-N-oxide | 0.216 | 0.214 | 0.218 | 0.219 | 0.211 | 0.210 | 0.215 | 1.7 |
DN-NFM Impurity | 0.216 | 0.218 | 0.215 | 0.215 | 0.216 | 0.212 | 0.215 | 0.9 |
DN-NFO Impurity | 0.198 | 0.195 | 0.213 | 0.192 | 0.214 | 0.215 | 0.205 | 5.2 |
GN | 0.203 | 0.212 | 0.204 | 0.206 | 0.205 | 0.210 | 0.207 | 1.7 |
DN | 0.182 | 0.189 | 0.194 | 0.188 | 0.187 | 0.201 | 0.190 | 3.4 |
Results of intermediate precision.
Compound | Prep-1 | Prep-1 | Prep-3 | Prep-4 | Prep-5 | Prep-6 | Avg. | % RSD |
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GN-Impurity A | 0.191 | 0.204 | 0.201 | 0.199 | 0.210 | 0.212 | 0.203 | 3.8 |
GN-Impurity B | 0.212 | 0.204 | 0.210 | 0.209 | 0.211 | 0.204 | 0.208 | 1.7 |
GN-Impurity C | 0.192 | 0.198 | 0.191 | 0.194 | 0.202 | 0.209 | 0.198 | 3.5 |
GN-Impurity D | 0.185 | 0.192 | 0.191 | 0.194 | 0.193 | 0.192 | 0.192 | 1.5 |
DN-Impurity A | 0.202 | 0.202 | 0.203 | 0.202 | 0.202 | 0.204 | 0.203 | 0.4 |
DN-Impurity B | 0.191 | 0.188 | 0.204 | 0.209 | 0.189 | 0.202 | 0.197 | 4.5 |
DN-Impurity C | 0.183 | 0.189 | 0.191 | 0.186 | 0.205 | 0.206 | 0.193 | 5.1 |
DN-N-oxide | 0.197 | 0.210 | 0.213 | 0.202 | 0.198 | 0.212 | 0.205 | 3.5 |
DN-NFM Impurity | 0.183 | 0.187 | 0.179 | 0.184 | 0.185 | 0.188 | 0.184 | 1.7 |
DN-NFO Impurity | 0.206 | 0.197 | 0.209 | 0.212 | 0.194 | 0.208 | 0.204 | 3.5 |
GN | 0.199 | 0.205 | 0.201 | 0.204 | 0.207 | 0.208 | 0.204 | 1.7 |
DN | 0.187 | 0.185 | 0.192 | 0.186 | 0.194 | 0.191 | 0.189 | 1.9 |
The determined limit of detection, limit of quantification, and precision at LOQ values for GN, DN, and their impurities were reported in Table
LOD, LOQ, and precision data.
Compound | LOD (%) | LOQ (%) | S/N ratio | % RSD at LOQ Level precision | |
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LOD | LOQ | ||||
GN-Impurity A | 0.004 | 0.001 | 2.7 | 9.5 | 4.2 |
GN-Impurity B | 0.008 | 0.002 | 2.9 | 9.8 | 2.5 |
GN-Impurity C | 0.005 | 0.002 | 3.0 | 9.8 | 3.1 |
GN-Impurity D | 0.003 | 0.001 | 2.9 | 9.7 | 2.5 |
DN-Impurity A | 0.014 | 0.004 | 2.8 | 10.1 | 1.1 |
DN-Impurity B | 0.021 | 0.006 | 2.7 | 10.4 | 3.4 |
DN-Impurity C | 0.023 | 0.007 | 2.8 | 9.9 | 4.9 |
DN-N-oxide | 0.016 | 0.005 | 3.1 | 9.8 | 2.2 |
DN-NFM Impurity | 0.016 | 0.005 | 2.7 | 10.1 | 1.5 |
DN-NFO Impurity | 0.041 | 0.012 | 2.9 | 10.6 | 4.2 |
GN | 0.006 | 0.002 | 3.1 | 9.8 | 2.1 |
DN | 0.015 | 0.005 | 2.8 | 9.6 | 2.8 |
The recovery of all the impurities from finished pharmaceutical dosage form ranged from 85.0% to 115.0%. The summary of % recovery for individual impurity was mentioned in Table
Accuracy of the method.
Compound | % Recovery at each levela | |||||
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LOQ | 0.1% | 0.2% | 0.4% | 0.8% | 1.0% | |
GN-Impurity A | 98.5 | 99.5 | 101.2 | 96.7 | 99.9 | 103.5 |
GN-Impurity B | 105.6 | 102.7 | 99.7 | 105.1 | 98.5 | 97.5 |
GN-Impurity C | 102.6 | 105.6 | 101.9 | 106.1 | 103.5 | 102.1 |
GN-Impurity D | 95.6 | 97.2 | 96.5 | 95.8 | 96.5 | 98.5 |
DN-Impurity A | 93.5 | 96.5 | 95.2 | 94.2 | 98.5 | 96.7 |
DN-Impurity B | 98.5 | 96.7 | 99.9 | 98.9 | 97.5 | 96.5 |
DN-Impurity C | 101.1 | 102.3 | 102.5 | 103.7 | 102.5 | 101.8 |
DN-N-oxide | 105.6 | 106.5 | 105.1 | 104.5 | 102.9 | 103.7 |
DN-NFM Impurity | 101.5 | 98.5 | 102.5 | 101.2 | 99.5 | 98.6 |
DN-NFO Impurity | 107.1 | 105.6 | 106.9 | 105.8 | 106.5 | 105.8 |
GN | 98.5 | 96.5 | 97.2 | 96.8 | 97.4 | 98.7 |
DN | 99.2 | 98.5 | 97.1 | 99.5 | 100.5 | 101.2 |
Linear calibration plot for the related substance method was obtained over the calibration ranges tested, that is, LOQ to 1.0% of the target test concentration. The correlation coefficient obtained was greater than 0.997 for all the components. The slope and y-intercept values were also provided in Table
Regression statistics.
Substance | Linearity range ( |
Correlation coefficient ( |
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GN-Impurity A | 0.93 to 239.61 | 0.999 | 0.9 |
GN-Impurity B | 2.01 to 242.78 | 0.999 | 1.0 |
GN-Impurity C | 1.26 to 241.92 | 1.000 | 0.7 |
GN-Impurity D | 0.79 to 242.55 | 0.999 | 0.8 |
DN-Impurity A | 0.17 to 14.10 | 1.000 | 0.4 |
DN-Impurity B | 0.25 to 10.10 | 1.000 | 0.1 |
DN-Impurity C | 0.27 to 12.23 | 1.000 | 0.8 |
DN-N-oxide | 0.19 to 12.49 | 1.000 | 0.6 |
DN-NFM Impurity | 0.19 to 12.19 | 0.998 | 1.5 |
DN-NFO Impurity | 0.48 to 12.22 | 1.000 | 0.9 |
GN | 1.53 to 244.78 | 0.999 | 1.6 |
DN | 0.18 to 12.15 | 1.000 | 0.1 |
(a) Linearity graph of Guaifenesin. (b) Linearity graph of Guaifenesin impurity A. (c) Linearity graph of Guaifenesin impurity B. (d) Linearity graph of Guaifenesin impurity C. (e) Linearity graph of Guaifenesin impurity D. (f) Linearity graph of Dextromethorphan. (g) Linearity graph of Dextromethorphan impurity A. (h) Linearity graph of Dextromethorphan impurity B. (i) Linearity graph of Dextromethorphan impurity C. (j) Linearity graph of Dextromethorphan N-oxide. (k) Linearity graph of Dextromethorphan impurity NFM. (l) Linearity graph of Dextromethorphan impurity NFO.
No significant effect was observed on system suitability parameters such as resolution, RSD, tailing factor, RRTs of impurities, or the theoretical plates of GN and DN when small but deliberate changes were made to chromatographic conditions. The results were presented in Tables
Results of robustness study.
S. no. | Impurity name | RRT’s of impurities | ||||||
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As such method | At 0.6 mL/min flow rate | At 1.0 mL/min flow rate | At 50°C column temperature | At 60°C column temperature | At pH 2.8 (buffer pH) | At pH 3.2 (buffer pH) | ||
1 | GN-Impurity A | 0.74 | 0.76 | 0.73 | 0.72 | 0.71 | 0.74 | 0.73 |
2 | GN-Impurity B | 1.12 | 1.11 | 1.13 | 1.11 | 1.10 | 1.12 | 1.13 |
3 | GN-Impurity C | 2.04 | 2.06 | 2.05 | 2.05 | 2.02 | 2.04 | 2.05 |
4 | GN-Impurity D | 2.14 | 2.12 | 2.15 | 2.13 | 2.11 | 2.13 | 2.14 |
5 | DN-Impurity B | 0.83 | 0.82 | 0.81 | 0.82 | 0.82 | 0.83 | 0.82 |
6 | DN-Impurity C | 0.98 | 0.97 | 0.98 | 0.98 | 0.97 | 0.97 | 0.98 |
7 | DN-N-oxide | 1.01 | 1.02 | 1.03 | 1.02 | 1.03 | 1.02 | 1.04 |
8 | DN-Impurity A | 1.03 | 1.04 | 1.05 | 1.04 | 1.05 | 1.04 | 1.06 |
9 | DN-NFM Impurity | 1.28 | 1.31 | 1.30 | 1.31 | 1.29 | 1.28 | 1.19 |
10 | DN-NFO Impurity | 1.38 | 1.41 | 1.42 | 1.39 | 1.39 | 1.39 | 1.41 |
Note: GN known impurities RRTs were calculated against GN main peak, and DN known impurities RRTs were calculated against DN main peak.
No significant changes were observed in the content of impurities during solution stability and mobile phase stability experiments when performed using the impurities method. The solution stability and mobile phase stability experiment data confirms that the sample solution and mobile phases used during the impurity determination were stable for at least 48 h.
The gradient HPLC method developed for the simultaneous determination of GN and DN impurities in pharmaceutical dosage form was precise, accurate, and specific. The method is validated as per ICH guidelines and found to be specific, precise, linear, accurate, rugged, and robust. The developed method can be used for the stability analysis of GN and DN either individually or in their combination dosage forms.
The authors wish to thank the management of Dr. Reddy’s group for supporting this work. The authors wish to acknowledge the formulation development group for providing the samples for their research. They would also like to thank colleagues in bulk manufacturers for providing chemicals and impurity standards for their research work.