Development and Validation of a Novel Stability-Indicating RP-HPLC Method for Simultaneous Determination of Tezacaftor and Ivacaftor in Fixed Dose Combination
Narendra Singh1,*, Parveen Bansal2, Mukesh Maithani2 and Yashpal Chauhan1
Abstract
Asimpleandprecisenovelstability-indicatingmethodforthesimultaneousestimationoftezacaftor and ivacaftor in combined tablet dosage form was developed and validated using reversed-phase high-performance liquid chromatography (RP-HPLC). The method is being reported for the first time and includes an estimation of degradation products produced post-stress conditions without any extraction or derivatization. The chromatographic separation of the drugs was achieved with a Symmetry Shield RP18 Column (100 Å, 5 µm, 4.6 mm × 250 mm) using a mixture of buffer, methanol and acetonitrile (42:27:31 v/v/v) as mobile phase. The buffer used in mobile phase contained 35 mM potassium dihydrogen phosphate, and its pH was adjusted to 7.0 ± 0.02 with 20% orthophosphoric acid. The instrument was set at flow rate of 1.2 mL min−1 at ambient temperature and the wavelength of UV-visible detector at 275 nm. The developed method could be suitable for the quantitative determination of these drugs in pharmaceutical preparations and also for quality controlinbulkmanufacturing.Stresstestingwasperformedtoprovethespecificity.Nointerference was observed from its stress degradation products. The statistical analysis was done by using F-test and t-test at 95% confidence level.
Introduction
Tezacaftor (TZ), 1-(2, 2-difluoro-1, 3-benzodioxol-5-yl)-N-[1-[(2R)2, 3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl) indol-5-yl] cyclopropane-1-carboxamide with molecular formula C26H27F3N2O6, is used as a corrector of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene function. Ivacaftor (IV) chemically N-(2, 4-Di-tert-butyl-5-hydroxyphenyl)-4oxo-1, 4-dihydroquinoline-3-carboxamide with molecular formula C24H28N2O3 is a CFTR potentiator approved for patients with the G551D mutation of CF, which account for 4–5% cases of CF (1–6). The clinical studies done with TZ–IV combination for the treatment of CF exhibited significant improvement in lung function (FEV1) of the patients. The FDA approved SYMDEKO (a combination formulation of TZ and IV) in 2018 for the treatment of CF in the patients aged 12 years or above, followed by the approval for the treatment of underlying cause of CF in children aged between 6 and 11 years in 2019. TZ moves the defective CFTR protein to the proper place in the airway cell surface, while IV facilitates the opening of the chloride channel on the cell surface to allow chloride and sodium (salt) to move in and out of the cell. SYMDEKO is used orally, and the recommended dose is one tablet (TZ 100 mg/IV 150 mg) taken in the morning and one tablet (IV 150 mg) taken in the evening,approximately 12 hours apart.It is taken with fat-containing food.
The literature survey revealed that there is no simple isocratic elution reversed-phase high-performance liquid chromatography (RP-HPLC)-based stability indicating method for the concomitant determination of TZ and IV in combination dosage form. Therefore, an attempt was made to develop a novel, simple, accurate and precise method for the simultaneous estimation of TZ and IV in combinedpharmaceuticaldosageform.Thismanuscriptdescribesthe development and validation of RP-HPLC method for simultaneous estimation of these drugs as per ICH guidelines (6–17).
Experimental
Chemicals and reagents
TZ and IV standards were obtained from Novartis India Ltd.(Maharashtra). Methanol, acetonitrile and acetic acid (HPLC grade) were procured from Central Drug House (P) Limited, New Delhi, India. Ammonium acetate AR, sodium dihydrogen phosphate AR and orthophosphoric acid AR grade were procured from Rankem, RFCL Limited, New Delhi, India. HPLC grade water was used throughout the study. Other chemicals used were of analytical or HPLC grade.
Instrumentation
The analysis was carried out on Waters Alliance e-2695 separating module (Waters Co., MA, USA) using photodiode array detector (waters 2998) with autosampler and column oven. The instrument was controlled by Empower Software (version 6.00.00.00) installed with equipment for data collection and acquisition. A Symmetry Shield RP18 Column (100 Å, 5 µm, 4.6 mm × 250 mm), eluted with mobile phase at the flow rate of 1.2 mL min−1, was used.
Chromatographic conditions
The mobile phase was consisted of a mixture of buffer, methanol and acetonitrile (42:27:31 v/v/v). The buffer contained 35 mM potassium dihydrogen phosphate. The pH of the buffer was adjusted to 7.0 ± 0.02 with 20% orthophosphoric acid. It was filtered through a 0.45 micrometer nylon filter and degassed in ultrasonic bath prior to use. All the measurements were made with injection volume 10 µL and UV detection at 275 nm.All analyses were performed at ambient temperature.
Standard and sample solution preparation
Standard stock solution. Standard solution was prepared by dissolving the drug in the solvent and diluting to the desired concentration. The mobile phase was used as solvent system. Accurately weighed 50 mg of TZ (99.23%) and IV (99.57%) were transferred into a 50 mL volumetric flasks separately and dissolved in the mobile phase. The volume was make up to the mark with mobile phase and mixed well.
Mixed standard solution. A mixed standard solution was prepared from these stock solutions by transferring 5 mL of each of the stock solution to a 50 mL volumetric flask and diluting with mobile phase to get a solution of 100 µg mL−1of each drug. Finally, seven different mix standard concentrations at a level of 10,20,50,80,100,120 and 150% (3.5,7.0,17.5,28.0,35.0,42.0 and 52.5µg mL−1,respectively) were prepared for both the drugs.
Preparation of sample solutions. The method was used for the quantitation of TZ and IV in the marketed tablet formulation (SYMDEKOTM tablets, Vertex Pharmaceuticals Incorporated). For sample preparation, the mobile phase was used as solvent. Twentyfive tablets were weighed and powdered finely. Tablet powder equivalent to 100 mg of TZ and 150 mg IV was transferred into 100 mL volumetric flask and dissolved in 25 mL of mobile phase.The volume wasmadeup tothemark.The solution wasultrasonicated for 30 minutes and filtered through a 0.45 micron membrane filter. The solution was further diluted with mobile phase to obtain the desired concentration (10 and 15 µg mL−1 of TZ and IV, respectively) and was subjected to HPLC analysis as described earlier. From the peak area of the chromatogram, the amount of drugs in samples was calculated.
Method validation
The optimized chromatographic conditions were validated by determining specificity, linearity, range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), robustness, ruggedness and system suitability parameters in accordance with the ICH guidelines Q2 (R1). To assess the linearity and range of the developed method, seven different mix standard concentrations at a level of 10, 20, 50, 80, 100, 120 and 150% (3.5, 7.0, 17.5, 28.0, 35.0, 42.0 and 52.5 µg mL−1, respectively) of TZ and IV were prepared. The analyses were performed in triplicate. The peak area values were plotted against the corresponding concentrations. Precision is the measure of the degree of repeatability of an analytical method under normal operation and is normally expressed as the %RSD for a statistically significant number of samples. There are two types of precision: repeatability and intermediate precision (ruggedness). The precision of the chromatographic method was estimated by measuring repeatability (intra-day assay precision) and intermediate precision (inter-day variation) for three consecutive days. The accuracy was measured by performing the assay of samples (spiked placebos) prepared at three concentration levels of 50, 100 and 150% of the standard concentration, with three replicates for each concentration. The %recovery and %RSD were calculated for each of the replicate samples.The LOD and the LOQ of the method were calculated based on the standard deviation of the response (σ) and slope approach as defined in the ICH guidelines. The LOD was calculated using the formula 3.3∗σ/slope, and the LOQ was calculated using the formula 10∗σ/slope. The robustness of the method was assessed under a variety of conditions including flow rate, pH and percentage of solvent in the mobile phase (18–21).
Forced degradation study
Forced degradation studies were performed to evaluate the stabilityindicating properties and specificity of the method.Intentional degradation was performed by exposing the formulation to five different stress conditions.The condition mentioned in Table I was followed in the stress study protocol.Stressed samples were analyzed periodically by checking the presence of related peaks and peak purity for the active ingredients (22–29).
Results
In this work a novel stability-indicating analytical LC method for the simultaneous determination of TZ and IV in bulk drug and pharmaceutical formulations with UV detection was developed and validated as per ICH guidelines for analytical method validation, Q2 (R1).
Method development
The main objective of this work was to develop a stability-indicating RP-HPLC method for the determination of TZ and IV within a short run time between 7 and 8 minutes and symmetry between 0.80 and 1.20. The method was developed by effecting systematic changes in the chromatographic conditions. The process involved the selection of appropriate conditions followed by optimization. The conditions included the type of column packing, column dimensions, mobile phase composition, flow rate, oven temperature, sample amount and detection wavelength. The stationary and mobile phases play an important role on theoretical plates, peak shape, symmetry and resolution. To obtain symmetrical peaks with better resolution and peak purity, various chromatographic conditions were investigated and optimized for the determination of TZ and IV, such as mobile phases with different composition, pH and stationary phases with different packing material, etc. In RP-HPLC, the pH of the eluent significantly influences the separation of the components. The pKa values of TZ and IV were calculated to establish the distribution of the conjugated acid/base species as a function of pH (30, 31). Furthermore,great care was taken in the preparation ofmobile phase, particularly the pH value adjustment as the pH of the mobile phase affects the resolution of nucleotides. Phosphate buffer with high pH was used to avoid problems like silica dissolution. Thus, different buffers having a pH between 6.0 and 7.5 were tried with different ratios of acetonitrile and methanol in isocratic condition. The best separation was achieved at pH 7.0. UV spectra of 10 µg mL−1 TZ and 10 µg mL−1 IV in mobile phase that were recorded by scanning in the wavelength range of 200 nm to 400 nm. The wavelength of 275 nm was noted to be the λmax as at this value both the drugs showed maximum absorbance. Both the drugs have high ratio of carbon to heteroatom and has conjugated bond; therefore, they can be separated best through C18 stationary phase mainly based on their overall hydrophobicity. In addition, TZ and IV can also be separated using phenyl–hexyl stationary phase considering their π electrons involving π–π interactions. Four kinds of HPLC columns, viz., SUPELCOSILTM LC-18-S HPLC Column, Hypersil ODS C18 Columns, Gemini C18 HPLC Columns and DiamonBondTM-C18 Columns, were tried with different mobile phase compositions. In these columns, broad characteristic peaks were obtained with all the ratios (30:70, 50:50, 60:20, 80:20) of methanol and water. No improvement of peak shapes was noted even when the temperature of column was increased to 40◦C.The number of theoretical plates with a mixture of methanol or acetonitrile with water as a mobile phase were below 1500 which indicated poor separation power. The peak symmetry and peak shape were also poor with the above two mobile phases which may be attributed to low polarity of the mobile phase. So phosphate buffer with different concentrations (30,35,40,45 and 50 mM) was used to improve polarity of the mobile phase, which resulted in a narrowed peak. However, the peak shape and peak symmetry were still not satisfactory. To improve this, acetonitrile with methanol was used. Finally, the mixture solution of phosphate buffer (35 mM), methanol and acetonitrile (42:27:31 v/v/v) was demonstrated to be the suitable mobile phase which gave best peak shape and peak symmetry. In addition to the composition of the mixture, the pH of the buffer was also found to be critical in the analyte separation and method optimization. The effect of the buffer pH on retention time was found to be related to the ionization of the solute. A series of mixture solutions with different pH values (6.0, 6.5, 7.0 and 7.5) was employed to investigate the retention time and resolution of the drugs keeping the other chromatographic parameters unchanged. A Waters Symmetry Shield RP18 Column (100 Å, 5 µm, 4.6 mm × 250 mm) was chosen for this study since it gave the highest efficiency with regard to the number of theoretical plates.
Finally, the mobile phase containing phosphate buffer (35 mM potassium dihydrogen phosphate), methanol and acetonitrile (42:27:31 v/v/v) (pH 7.0 ± 0.02 adjusted with 20% orthophosphoric acid) was selected and found to be optimal with more theoretical plates (≥12,500), narrow peak, high peak symmetry (0.98 and 1.15) and short retention time (3.75 and 6.04 minutes, below 8 minutes). Based on the optimal mobile phase, highly symmetrical and sharp characteristic peaks of the drugs were further obtained on Symmetry Shield RP18 Column (100 Å, 5 µm, 4.6 mm × 250 mm) with 1.2 ml min−1 flow rate. A typical HPLC chromatogram obtained during simultaneous determination of TZ and IV is given in Figure 2.
Method validation
An optimized method must be validated before actual use. System suitability testing was performed as per ICH guidelines for analytical method validation, Q2 (R1).
Specificity
The specificity studies proved the absence of interference, since no other peak appeared at the retention times of TZ and IV (3.75 and 6.04 minutes). Moreover, interaction studies indicated that the analytes did not interact with each other and were well within the acceptance level of 2.0% RSD.
Range and linearity
Seven different concentrations levels of 10, 20, 50, 80, 100, 120 and 150% (3.5, 7.0, 17.5, 28.0, 35.0, 42.0 and 52.5 µg mL−1, respectively) of the mixture of TZ and IV were prepared for linearity studies. The calibration curves obtained by plotting the peak area against the concentration showed linear relationship. Calibration curves with corresponding residual plots of TZ and IV are shown in Figure 3. The linear regression equations for TZ and IV were found to be y = 29,376 x + 2,243 and y = 24,008 x + 13,776, respectively. The regression coefficient (R2) values for TZ and IV were noted to be 0.999 and 0.997, respectively. The results showed an excellent correlation between the peak area and concentration of drug within the selected concentration range. The results confirmed the linearity and the reproducibility of the assay method. The F-test and Student’s t-test (P < 0.05) confirmed insignificant difference in the predicted and observed values. The regression characteristics of the proposed HPLC method are given in Table II.
Precision (repeatability and intermediate precision)
The precision of an analytical method is the extent of agreement between individual test results when the method is applied repetitively to multiple sampling of homologous sample. The precision of the method was evaluated by analyzing assay of six different samples (Table III). The %RSD values of the assay from the six different samples were found to be 0.39 and 0.29% for TZ and IV, respectively. The ruggedness (intermediate precision) of the method was determined using another system and column for the analysis in a different day. The low value of %RSD (1.02 and 1.18% for TZ and IV, respectively) showed that the method is precise within the acceptance limit of 2%. The intra- and inter-day variability or precision data are given in Table IV. The results indicated good precision of the developed method. The results obtained from the intra- and inter-day were evaluated statistically using the F-test and Student’s t-test. The calculated value of F-test and Student’s t-test indicated that the intra- and inter-day data did not differ significantly in terms of precision (Table IV).
Accuracy
Therecoveryexperiments wereperformed byaddingknownamounts of the drugs in the placebo at three levels, 50, 100 and 150% of the label claim of the marketed formulation. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The mean recovery values were found to be 99.23 and 101.39%. The results revealed that there was no interference of excipients. The results of accuracy are shown in Table V.
Limit of detection and limit of quantitation
The LOD and LOQ predict the sensitivity of the method. The LOD for TZ and IV were found to be 0.18 and 0.28 µg mL−1, respectively, whereas LOQ were noted to be 0.51 and 0.77 µg mL−1, respectively. The values indicated that the method is sensitive.The chromatograms are shown in Figure 4.
Robustness
The robustness of a method is the ability to remain unaffected by small changes in parameters. The experimental conditions were purposely altered,and the chromatographic resolution of TZ and IV was assessed. To study the effect of the organic solvent (acetonitrile and methanol) composition on resolution,the concentration was changed 2 units on either side, while other chromatographic conditions were kept constant. To study the effect of flow rate on resolution, it was changedto0.1unitoneithersidefrom1.2to1.1mLmin−1and1.3mL min−1, while other conditions were held constant. To study the effect of pH on resolution, it was changed to ±0.3 unit on either side from 7.0 to 7.3 and 6.7, while other conditions were held constant. The resolution between TZ and IV was not less than 1.5 in the study. The results are shown in Table VI.
Analysis of tablet formulations
The developed method was successfully applied to analyze TZ and IV in marketed tablet formulation. The amounts recovered were expressed as percentage of the label claim. The analysis of the marketed tablets (SYMDEKOTM tablets, Vertex Pharmaceuticals Incorporated) was carried out using an optimized mobile phase and HPLC conditions. The average percentage of drug contents of tablets obtained by the developed method for TZ and IV was noted to be 100.18% and 99.26%, respectively. A typical HPLC chromatogram of a tablet sample solution containing TZ and IV is shown in Figure 5a. These values comply with the official specifications as ±5% of the label claim (i.e. 95.0–105.0%).
Forced degradation study
All the stress conditions applied were enough to degrade TZ and IV in the pharmaceutical formulation. TZ and IV were degraded and remained ∼82.22 and ∼84.41% respectively,when 2 N hydrochloric acid was used at 75◦C for 5 hours. TZ and IV were degraded and remained ∼90.18 and ∼94.87%, respectively, when 2 N sodium hydroxide solution was used at 75◦C for 5 hours. TZ and IV were degraded and remained ∼71.65 and ∼79.94%, respectively, under 10% hydrogen peroxide solution at 75◦C for 5 hours. TZ and IV were degraded and remained ∼97.88 and ∼98.58%, respectively, under 75◦C for 48 hours. TZ and IV were degraded and remained ∼98.49 and ∼98.78%, respectively, under overall illumination of ≥210 Wh/m2 at 25◦C for 7 days with UV radiation at 320–400 nm. From these stress studies, it was thus concluded that TZ and IV were not stable in strong basic, strong acidic and oxidative conditions but stable in thermal and photolytic conditions, and developed method can be considered highly specific for intended use.
The chromatograms of stress studies of TZ and IV are given in Figure 5b–f. Peak purity was verified for all the stressed sample chromatograms by using the PDA detector. It is the weighted average of all spectral contrast angle calculated by comparing the spectra from all data points in the integrated peak against the peak apex spectrum. The peak that showed the purity angle less than the purity threshold indicated the purity of the peak, i.e. no other peaks coeluted with that peak. If the purity angle is greater than the purity threshold, there is something within the peak and the peak is not pure. The obtained purity angle and purity threshold limits confirm that peaks are pure and homogenous in all stressed conditions. The results of stress studies of TZ and IV are shown in Tables VII and VIII, respectively.
System suitability parameters
For system suitability parameters, six replicates of mixed standard solution were injected. All critical parameters met the acceptance criteria on all days. Parameters such as resolution, tailing factor, theoretical plate, capacity factor, retention volume and asymmetry factor of the peaks were calculated. The results are shown in Table IX.
Discussion
The developed method is the first stability-indicating analytical method for the determination of TZ and IV under varied stress conditions. Good chromatographic resolution was obtained using a Symmetry Shield RP18 Column (100 Å, 5 µm, 4.6 mm × 250 mm) stationaryphasewithanisocratic elutionofamobilephaseconsisting of buffer, methanol and acetonitrile (42:27:31 v/v/v) at a flow rate of 1.2 mL min−1 and UV detection at 275 nm. The analytical method was appropriately validated in terms of system suitability, linearity, LOD,LOQ,specificity,accuracy,repeatability,intermediate precision and robustness tests. The method exhibited good linearity in the concentration range of 3.5–52.5 µg mL−1. The regression coefficient values indicated excellent degree of linearity when the peak area was used for signal evaluation. A series of dilutions of the standard stock solution were made to determine the detection and quantification limits based on the signal-to-noise ratios.
The method could be very useful for the qualitative and quantitative analysis of TZ and IV and their degradation products under various stress conditions (basic, acidic, oxidative conditions, thermal and photolytic). The method carries the advantage of being more reproducible. The method seems quite suitable for the concomitant analysis of the drugs in marketed formulations as no interference of the excipients was noted.The results of the recovery studies exhibited high degree of accuracy of the method with an added advantage of being economic. The method is extremely simple without any complex procedures. The method showed high selectivity, precision and reproducibility. These observations suggest that the method could be successfully applied for the routine analysis of the drugs either in their pure bulk powders or in their dosage forms without the requirement of prior separation.
Conclusion
A simple and accurate RP-HPLC method for simultaneous estimation of TZ and IV was developed and validated as per ICH guidelines. The developed method is a novel simple isocratic elution protocol based on RP-HPLC method with additional advantage of simple sample treatment and short resolution time. It is simple, precise (RSD < 2%) and with high recovery (more than 98%). The method was successfully validated as per ICH guidelines for the analysis of bulk drugs and dosage forms. The method was also found to be robust with respect to pH, flow rate and composition of mobile phase. In addition, simple easy extraction procedure and isocratic elution offered rapid and cost-effective analysis of the drugs. The statistical treatment proved the validity of the method. The method can be used for the routine analysis of TZ and IV in combined dosage form and in the quality control in bulk manufacturing as well. The developed method is also qualified and reliable to demonstrate and detect any change in the drug product assay during stability studies.
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