Development and validation of high performance liquid chromatography method for the determination of nifedipine in drug - excipient compatibility testing samples

To develop and validate the determination method of nifedipine in drug - excipient compatibility testing samples by high performance liquid chromatography.

Journal of military pharmaco-medicine no9-2019 DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE DETERMINATION OF NIFEDIPINE IN DRUG - EXCIPIENT COMPATIBILITY TESTING SAMPLES Tran Quang Trung1; Nguyen Thanh Hai2; Trinh Van Lau3; Truong Ngoc Hien1 SUMMARY Objectives: To develop and validate the determination method of nifedipine in drug excipient compatibility testing samples by high performance liquid chromatography Methods: Chromatographic conditions include: PDA detector, analytical wavelength, column and mobile phase were studied; the evaluation of the system suitability, specificity - selectivity, the linearity range, repeatability, intermediate precision, accuracy, range, limit of detection and limit of quantitation following the guidance of ICH Results: Chromatographic conditions were established, including: PDA detector, analytical wavelength 237 nm, column RP-C18 Phenomenex, mobile phase MeOH:H2O (65/35, v/v) The method had been evaluated and achieved the ICH’s regulations Conclusion: The developed HPLC method can be used to determine the nifedipine in drug - excipient compatibility testing samples, which will use in pre-formulation studies of the nifedipine dosage forms * Keywords: Nifedipine; High performance liquid chromatography; Drug - excipient compatibility INTRODUCTION Nifedipine (NIF), a dihydropyridine calcium channel blocker, is widely used in the treatment of angina, hypertension and other vascular disorders [2, 3] However, nifedipine is relatively unstable [4] and can interact with excipients in tablets formulation that reduce its content In order to determine the interaction between NIF and excipients to select suitable excipients for formulation development of NIF push - pull osmotic pump extendedreleased tablets, we have developed and validated the HPLC method to quantify NIF in drug - excipient compatibility testing samples created by combining NIF with potential excipients Vietnam Military Medical University Vietnam National University, Hanoi National Institute of Drug Quality Control Corresponding author: Tran Quang Trung (tqt201316@gmail.com) Date received: 05/10/2019 Date accepted: 21/11/2019 163 Journal of military pharmaco-medicine no9-2019 MATERIALS AND METHODS Materials and equipment * Materials and chemicals: - Reference standard of NIF (potency 99.82%) was provided by National Institute of Drug Quality Control - NIF was supplied by Baoji Guokang Bio-Technology Co., Ltd (China) and meet USP 38 standards - PEO (polyethylene oxide) N10, PEO N80, PEO N750, PEO 301, PEO 303, PEO Coagulant provided by Colorcon Asia Pacific Pte., Ltd - Lactose monohydrate, sodium chloride, magnesium stearate, PVP K30, red iron oxide were sourced from China and all meet USP 38 standards - HPLC grade acetonitrile, methanol were procured from Merck Ltd - Other used chemicals were of HPLC or analytical grade * Equipment: HPLC system Alliance Waters 2695D; Detector 2998 dual α Absorbance (USA); Phenomenex LUNA column, Xbridge column (RP-C18, µm, 250 x 4.6 mm); Spectrometer EMC-61PC-UV, Emclab (Germany); pH meter Mettler Toledo (Switzerland); analytical balance Mettler Toledo (precision 0.1 mg), other usually equipment in laboratory Methods * Method development: - Preparation for standard solutions: + Standard stock solution: A standard stock solution containing 500 µg/mL NIF was prepared by exactly approximately weighing 50.0 mg NIF standard, 164 transferring it into a 100 mL volumetric flask and adding about 70 mL mobile phase composed of methanol/H2O (65/35, v/v) The flask was sonicated for 30 mins, allowed to cool at room temperature and the volume was made up with mobile phase + Standard solution: Accurately taken mL of the stock standard solution 500 µg/mL into a 10 mL volumetric flask and made up the volume with mobile phase, mixed well to give a standard solution of about 150 µg/mL of NIF All standard solutions were filtered through a 0.45 µm PTFE membrane filter prior to injection into the HPLC system - Preparation for sample solutions: Accurately weighed an amount of powder mixture of NIF and excipients (in a ratio of 1/1) equivalent to about 50.0 mg of NIF, transfered into a 100 mL volumetric flask; 70 mL mobile phase (methanol/H2O, 65/35, v/v) was added, and the flask was sonicated for 30 mins, allowed to cool at room temperature and made up the volume with mobile phase From this solution, a volume of about 10 mL of solution was withdrawn and was centrifuged at 4,000 rpm for 20 minutes Taken exactly mL of centrifuged solution into a 10 mL volumetric flask and add the mobile phase just to the mark, mixed well, filtered through a 0.45 µm PTFE membrane filter prior to injection into the HPLC system - Chromatographic conditions: The NIF standard solution of 150 µg/mL concentration was used for investigation of chromatographic conditions The solutions were scanned under 200 - 500 nm wavelength range to find the maximum Journal of military pharmaco-medicine no9-2019 absorption wavelength The other chromatographic conditions such as: stationary phase (silica gel column C18 Phenomenex LUNA, Xbridge); mobile phase (mixture of solvents MeOH, ACN, H2O at different proportions) were also studied Maintained a flow rate at mL/min, sample injection volume of 25 µL Identified chromatographic conditions for peaks with good symmetry, max peak area/concentration ratio and peak height/concentration ratio * Method validation: The proposed HPLC-UV method was validated in accordance with the International Conference on Harmonization (ICH) guidelines and Circular 32/2018 of the Vietnamese Ministry of Health [1,5], by evaluating the validation characteristics such as the system suitability, specificity selectivity, the linearity range, precision (repeatability, intermediate precision), accuracy, specified range, limit of detection and limit of quantitation RESULTS AND DISSCUSION Method development results * Analytical wavelength: The appropriate wavelength for NIF analysis was determined by recording UV spectrum of a NIF standard sample of 150 µg/mL on the EMC-61PC-UV spectrophotometer, Emclab The solution above was scanned from 200 to 500 nm with quartz cuvettes cm of thick The blank sample was mobile phase composed of methanol/H2O (65/35, v/v) Figure 1: Ultraviolet absorption spectrum of NIF standard solution of 150 µg/mL UV-Vis spectrum showed that the absorption maxima of NIF was 237 nm and 370 nm From there, choose the wavelength 237 nm as the wavelength for NIF qualification by HPLC method 165 Journal of military pharmaco-medicine no9-2019 * Column efficiency validation: Analyzed samples on two reversed silicagel columns C18 Xbridge and Phenomenex LUNA Table 1: Column analysis validation No Results Column Asymmetry factor (AF) Theoretical plate (N) Retention time (min) Phenomenex LUNA 1.120 10600 8.965 Xbridge 1.049 19385 13.108 Figure 2: Chromatogram of the NIF standard sample using Xbridge column Figure 3: Chromatogram of the NIF standard sample using Phenomenex LUNA column The results of asymmetry factor, the number of theoretical plates, retention time and chromatograms showed that both columns had a symetrical peak, the AF was within the permitted limits (0.8 ≤ AF ≤ 1.5); however, the Phenomenex LUNA column with a smaller number of theoretical plates, shorter retention time compared to these ones in the Xbridge column (13.108 mins), therefore, the Phenomenex LUNA column was selected for the quantitative analysis of NIF 166 Journal of military pharmaco-medicine no9-2019 * Mobile phase: Based on the reference documents and chemicals, solvents available in laboratory, three different mobile phase systems were studied including: - System 1: MeOH:H2O (65/35, v/v) - System 2: ACN:H2O (65/35, v/v) - System 3: ACN:MeOH:H2O (25/25/50, v/v/v) Table 2: Mobile phase investigation results Mobile phase Retention time (min) Asymmetry factor (AF) MeOH:H2O (65/35, v/v) 8.965 1.120 ACN:H2O (65/35, v/v) 5.187 1.261 ACN:MeOH:H2O (25/25/50, v/v/v) 1.050 1.040 Figure 4: Chromatogram of standard sample in mobile phase system MeOH:H2O (65/35, v/v) Figure 5: Chromatogram of standard sample in mobile phase system ACN:H2O (65/35, v/v) 167 Journal of military pharmaco-medicine no9-2019 Figure 6: Chromatogram of standard sample in mobile phase system ACN:MeOH:H2O (25/25/50, v/v) Chromatogram of standard sample in system included ACN:H2O (65/35, v/v) had unbalanced peak with AF = 1.261 and the baseline was not stable That one in system composed of ACN:MeOH:H2O (25/25/50, v/v/v) had balanced peak but retention time was too short (1.050 minutes) Whereas, this one in system including MeOH:H2O (65/35, v/v) had stable baseline, sharp and proportioned peak with AF = 1.126, indicated good separation ability * Mobile phase ratio: The mobile phase system which composed of mixture of MeOH and H2O was investigated at different ratio 50/50, 65/35, 60/40, 70/30, 80/20 Table 3: Results of investigation mobile phase ratio MeOH:H2O (v/v) Retetion time (min) Speak (µV.s) Asymmetry factor (AF) 50/50 16.328 2500234 1.059 65/35 8.981 12823315 1.127 60/40 13.315 12833722 1.091 70/30 6.601 12894973 1.169 80/20 4.261 12810176 1.258 Figure 7: NIF standard chromatogram with mobile phase MeOH:H2O (50:50, v/v) 168 Journal of military pharmaco-medicine no9-2019 Figure 8: NIF standard chromatogram with mobile phase MeOH:H2O (65:35, v/v) Figure 9: NIF standard chromatogram with mobile phase MeOH:H2O (60:40, v/v) Figure 10: NIF standard chromatogram with mobile phase MeOH:H2O (70:30, v/v) Figure 11: NIF standard chromatogram with mobile phase MeOH:H2O (80:20, v/v) 169 Journal of military pharmaco-medicine no9-2019 It was found that the mobile phase with the ratio of MeOH:H2O (65:35, v/v) was selected for quantitative analysis of NIF, because of good separation with a suitable retention time, compact and balanced peak Method validation results * System suitability test: Replicated the injection of a NIF standard solution at concentration of about 150 µg/mL times on HPLC system Evaluated retention time (RT), peak area and asymmetry factor and theoretical plate (N) in all chromatograms Table 4: Results of investigation system suitability Sample RT (min) Speak (µV.s) AF N 8.661 12287964 1.138 10873 8.718 12303091 1.137 10833 8.768 12318713 1.136 10814 8.794 12270688 1.136 10809 8.803 12260808 1.135 10795 8.795 12271121 1.136 10789 Mean 8.757 12285398 1.136 10819 RSD (%) 0.6 0.2 0.1 0.3 The results showed that %RSD for the retention time, peak area, asymmetry factor and theoretical plate of NIF standard were less than 2.0% The obtained value demonstrated the suitability of the system for the analysis of NIF * Specificity: Prepared samples: blank sample (mixture of excipients), standard sample, test sample Sample processing and chromatography carried out under selected conditions Figure 12: Chromatogram of blank sample 170 Journal of military pharmaco-medicine no9-2019 Figure 13: Chromatogram of NIF standard solution at concentration of 150 µg/mL Figure 14: Chromatogram of NIF test sample at concentration of 150 µg/mL Standard sample peak was balanced, retention time was 8.874 minutes On the chromatogram of the blank sample, no peak was observed in a period of - 10 minutes The chromatograms of both the test sample and the standard sample had peak at approximately 8.8 minutes From there, we concluded that the method was highly specific * Calibration curve and linearity range: Standard solutions were preparedat concentrations from 30 µg/mL to 240 µg/mL Each concentration was analyzed times Record chromatograms and peak response The relationship between the area and concentration of NIF was studied Table 5: Results of calibration curve (n = 3) Sample Conc (µg/ml) Speak (µV.s) Sample Conc (µg/ml) Speak (µV.s) 30 2233484.5 ± 12920.5 150 12282186.4 ± 5241.4 60 4757893.5 ± 3141.6 180 14819683.6 ± 17694.6 90 7274879.5 ± 8018.9 210 17343338.7 ± 35401.3 120 9786676.6 ± 12100.6 240 19868707.9 ± 28642 Regression equation: y = 83930x - 284700 Correlation coefficient: R = 171 Journal of military pharmaco-medicine no9-2019 The correlation coefficient R2 = indicated that a linear correlation between the concentration of NIF and the peak area was obtained in the range of concentrations investigated This linear range was suitable for quantifying NIF in drug - excipient compatibility testing samples * Precision: - Repeatability: test samples were prepared according to the selected procedure and injected into the HPLC system (prepared by tester 1) Determined the precision by calculating the relative standard deviation between the quantifications Table 6: Results of method precision No Weight (g) Speak (µV.s) Content (%) 0.6450 13239283 100.43 0.6560 13373746 99.75 0.6540 13291701 99.44 0.6528 13280630 99.54 0.6545 13349152 99.79 0.6520 Mean 13277960 99.64 13302079 RSD% = 0.53 Under the selected chromatographic conditions, RSD of the quantitative results in each day of analysis wss 0.53%, which was less than 2% As such, the chosen method guarantees accuracy - Intermediate precision: test samples were prepared by another tester (tester 2) following the selected procedure and injected them into the HPLC system Determined the intermediate precision by calculating the relative standard deviation between the quantifications Table 7: Results of intermediate precision No Weight (mg) Speak (µV.s) Content (%) 0.6547 13366813 99.77 0.6565 13424439 99.92 0.6555 13451567 100.28 0.6534 13354395 99.87 0.6550 13420425 100.13 0.6538 13330146 99.63 Mean RSD% = 0.24% Under the selected chromatographic conditions, RSD of the quantitative results was analyzed by each analyst was 0.24% and 0.53%, all of them were less than 2% 172 Journal of military pharmaco-medicine no9-2019 By both of analysts were 0.3% which was less than 3% As such, the chosen method guarantees precision * Accuracy: The accuracy of the method expressed in % recovery % recovery was determined by calculating the amounts of standard added into the placebo powder at three different concentration levels (80%, 100%, 120%) Each level was made in triplicate (n = 3) Table 8: Results of accuracy test Recovery level Amount of the standard added (mg) Total peak area (µV.s) Amount of the standard recovered (mg) % recovery 41.8 10211936 41.5 99.28 41.4 10140050 41.2 99.52 42.0 10212840 41.5 98.81 51.9 12564631 51.1 98.46 51.3 12521695 50.9 99.22 51.7 12558812 51.1 98.84 60.5 14916732 60.7 100.33 61.2 14985984 60.9 99.51 61.8 15066105 61.3 99.19 Results Mean = 99.2 80% RSD = 0.36% Mean = 98.84 100% RSD = 0.38% Mean = 99.68 120% RSD = 0.59% The amount of standard recovery at each concentration level was in the range of 98 - 102% compared to the amount of standard added, with the RSD of % recovery at each concentration level < 2% indicated that the proposed method was highly accurate * Specified range: The accuracy results showed that at the NIF concentration of about 80% to 120% compared to the quantitative concentration, the quantitation method achieved good accuracy and repeatability Thus, the specified range of the method was 120 - 180 µg/mL * Determination of limit of determination and limit of quantification: The limit of detection and limit of quantification were estimated from signal to noise ratio Limit of detection and limit of quantification were found to be 0.0375 µg/ml and 0.105 µg/mL, respectively 173 Journal of military pharmaco-medicine no9-2019 CONCLUSION A HPLC method has been developed to quantify NIF in drug - excipient compatibility testing samples with chromatographic conditions such as: HPLC system Alliance Waters 2695D; detector 2998 dual α Absorbance; column (RP-C18, µm, 100A0, 250 x 4.6 mm) Phenomenex LUNA; mobile phase MeOH:H2 O (65/35, v/v); flow rate: mL/min; detector PDA 237 nm; injection volume 25 µL; column temperature: room temperature The proposed HPLC method was validated by evaluating the validation characteristics such as system suitability, specificity - selectivity, the linearity range, repeatability, intermediate precision, accuracy, range, limit of detection and limit of quantitation The analytical method had met the requirements according to 174 ICH regulations and Circular 32/2018 of the Vietnam Ministry of Health This method can be used to quantify NIF in drug - excipient compatibility testing samples REFERENCES Vietnamese Ministry of Health Circular 32/2018, Regulate the registration of drugs and drug materials Uday Y.A et al Estimation of nifedipine by reverse phase high performance liquid chromatography tablet dosage form IJPLS 2011, pp.610-612 Sweetman S.C et al Martindale Pharmaceutical Press 2014, pp.1447-1455 Klaus Florey et al Analytical profiles of drug substances Academic Press InC 1989, Vol 18, pp.245-253 ICH Harmonised Tripartite Guideline Guidelines for validation of analytical procedures: Q2 (R1) Text and Methodology 2005  ... NIF and the peak area was obtained in the range of concentrations investigated This linear range was suitable for quantifying NIF in drug - excipient compatibility testing samples * Precision: -. .. concentration, the quantitation method achieved good accuracy and repeatability Thus, the specified range of the method was 120 - 180 µg/mL * Determination of limit of determination and limit of quantification:... NIF in drug - excipient compatibility testing samples REFERENCES Vietnamese Ministry of Health Circular 32/2018, Regulate the registration of drugs and drug materials Uday Y.A et al Estimation of