|Year : 2018 | Volume
| Issue : 4 | Page : 124-129
Development and validation of simple simultaneous analysis for amlodipine and glibenclamide by nonderivatization high-performance liquid chromatography-fluorescence
Febrina Amelia Saputri1, Anisahtul Alawiyah1, Ayu Brilliany Firsty1, Sandra Megantara1, Arif Satria Wira Kusuma2, Taofik Rusdiana3, Aliya Nur Hasanah1, Mutakin Mutakin1, Ingrid S Surono4, Rizky Abdulah5
1 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia
2 Department of Biological Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia
3 Department of Pharmaceutical and Formulation Technology, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia
4 Department of Food Technology, Faculty of Engineering, Bina Nusantara University, Indonesia
5 Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia
|Date of Web Publication||10-Dec-2018|
Mrs. Febrina Amelia Saputri
Jl. Raya Bandung Sumedang, Km. 21, Jatinangor, Sumedang 45363
Source of Support: None, Conflict of Interest: None
Studies have shown that about 65% of diabetics have hypertension. Treatment for diabetic patients with hypertension is usually given a combination of drugs such as amlodipine (AML) and glibenclamide (GLI). The aim of this study was to develop and validate the simple simultaneous analysis method for separation of AML and GLI using high-performance liquid chromatography (HPLC) with fluorescence detector without derivatization. The arrangement of isocratic and gradient methods, mobile phase compositions, and flow rates to develop and validate the simple simultaneous analysis method for separation of AML and GLI by nonderivatization HPLC fluorescence was done. Optimum condition was obtained using an RP 18 (125 mm × 4 mm, i.d., 5 μm) and guard column RP 18 (4 mm × 4 mm, i.d., 5 μm) with mobile phase composition containing acetonitrile and phosphate buffer pH 3.0 using a 20:80 gradient condition at flow rate 1.0 ml/min measured at 361 nm for λ excitation and 442 nm for λ emission for AML and 235 nm for λ excitation and 354 nm for λ emission for GLI. The analysis of AML and GLI demonstrated a valid result with r2 value 0.999, recoveries were 100.04% and 99.14% relative standard deviations were 0.508% and 0,797%, respectively, detection limits were 0.055 and 0.104 μg/ml, and quantification limits were 0.166 and 0.316 μg/ml, respectively. An accurate method of separation for AML and GLI using HPLC with fluorescence detector without derivatization has been validated.
Keywords: Amlodipine, glibenclamide, high-performance liquid chromatography-fluorescence, nonderivatization, simultaneous
|How to cite this article:|
Saputri FA, Alawiyah A, Firsty AB, Megantara S, Wira Kusuma AS, Rusdiana T, Hasanah AN, Mutakin M, Surono IS, Abdulah R. Development and validation of simple simultaneous analysis for amlodipine and glibenclamide by nonderivatization high-performance liquid chromatography-fluorescence. J Adv Pharm Technol Res 2018;9:124-9
|How to cite this URL:|
Saputri FA, Alawiyah A, Firsty AB, Megantara S, Wira Kusuma AS, Rusdiana T, Hasanah AN, Mutakin M, Surono IS, Abdulah R. Development and validation of simple simultaneous analysis for amlodipine and glibenclamide by nonderivatization high-performance liquid chromatography-fluorescence. J Adv Pharm Technol Res [serial online] 2018 [cited 2021 Aug 2];9:124-9. Available from: https://www.japtr.org/text.asp?2018/9/4/124/247173
| Introduction|| |
Diabetes mellitus is a disorder characterized by elevated blood sugar levels or chronic hyperglycemia. Some of the body's metabolism can trigger the occurrence of diabetes if there is interference metabolism of fats, carbohydrates, and proteins and can also be exacerbated by vascular disorders. Diabetes is usually accompanied by various complications, such as increased kidney disorders, heart attacks, and stroke. Research has shown that about 65% of diabetic patients are usually accompanied by hypertension. And it has been proven that cardiovascular disorders can lead to morbidity and mortality in diabetic patients.
Amlodipine (AML) is a dihydropyridine derivative with a working mechanism inhibiting calcium ion channels that can be used for the treatment of antihypertensive and angina pectoris. It inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle, thereby reducing blood pressure and increasing blood flow to the heart muscle. The molecular weight of AML is 408.879 g/mol. AML has two hydrogen bonds donor and seven hydrogen bonds acceptor. It has logP of 3 and 75.3 mg/L solubility in water.
Glibenclamide (GLI), 1-(4-[2-(5-chloro-2-methoxybenzamido) ethyl] benzenesulfonyl)-3-cyclohexylurea, is a second-generation sulfonylurea hypoglycemic agent that appears to lower blood glucose by stimulating the release of insulin from the pancreas. The logP of GLI is 4.7, with the solubility in water being 4 mg/L. The molecular weight of GLI is 494.003 g/mol. It has three hydrogen bonds donor and five hydrogen bonds acceptor.
Simultaneous use of AML and GLI for the treatment of diabetics accompanied by hypertension resulted in good treatment outcomes characterized by decreased blood glucose, total cholesterol, creatinine urine, and creatinine clearance significantly. Interaction of the use of both drugs provides a higher percentage of inhibition small compared to the percentage of drug combination inhibition between GLI and enalapril.
AML is a fluorescent compound as well as fluorescence GLI that can be analyzed using high-performance liquid chromatography (HPLC) fluorescence., The advantages of fluorescence detector are the time required to be rapid and at low cost and can increase the detection and selectivity limits of a compound in the analysis.,
The use of a fluorescence detector against a spectrophotometric instrument has been performed and provides a more sensitive detection limit when compared to the use of an ultraviolet (UV) detector of 0.05 μg/ml. The use of an HPLC instrument with a UV detector to analyze the compound has been carried out by a more sensitive detection limit value than the use of the spectroscopy instrument of 2 ng/ml, but the lack of this method is the presence of peak interference that may interfere with the results of the analysis. The analytical method using HPLC without derivatization is also capable of producing a low detection limit value of 50 pg/ml, 0.18 ng/ml, and 0.4 ng/ml.,,
The purpose of this study was to validate the optimum conditions for the simultaneous separation of AML and GLI using HPLC without derivatization process so that this method of analysis could be used as a simple and validated alternative method for the determination of AML and GLI.
| Materials and Methods|| |
AML besylate and GLI standards were purchased from Sigma Aldrich, NaH2 PO4 and Na2 HPO4 pro analysis were purchased from Merck, acetonitrile and methanol HPLC grade were purchased from Merck, and aqua bidestillata was purchased from IPHA Laboratories.
An HPLC System (Waters e2695 Separation Module from Waters Associates, Milford, Massachusetts, USA) with the fluorescence detector (Waters Separation 2475 FLR Detector from Waters Associates) was used. The data were recorded using Empower Software from Waters Associates. The mobile phase used the percentage of acetonitrile with buffer phosphate. Stationary phase used was LiChrospher RP 18 (Merck, Darmstadt, Germany) (125 mm × 4 mm, i.d., 5 μm) and guard column Lichrospher RP 18 (Merck) (4 mm × 4 mm, i.d., 5 μm).
Preparation of standard stock solution
A total of 5 mg AML besylate and GLI were separately weighed and dissolved in 50 ml methanol HPLC grade. The standard solution concentration of 100 μg/ml was obtained. The standard stock solution of each solution was prepared to be a mixture of AML and GLI solutions, resulting in a mixed concentration of 0.1, 0.5, 1, 5, and 10 μg/ml, sonicated for 5 min and cooled to room temperature, mixed well, and filtered through 0.45 μm Millipore filter. We injected separately 10 μl of the standard solution into the equilibrated HPLC system in five replicates and measured the response of the major peak due to AML besylate and GLI.
System suitability testing of high-performance liquid chromatography
The system suitability of HPLC was carried out by gradient elution method and isocratic elution method using the mixed solution of 10 μg/ml which then calculated some parameters such as resolution, capacity factor, selectivity factor, tailing factor, theoretical plate, and height equivalent to a theoretical plate. The conditions of HPLC are shown in [Table 1].
Validation of the method
Validation of analytical method was done using mixed AML and GLI solution, and values of some parameters such as accuracy, precision, linearity, specificity, detection limit, quantification limit, and robustness were validated.
| Results|| |
The system suitability test
Before performing the validation process, the system suitability test is necessary because the results of this test will affect accuracy and precision. The system suitability test was performed using two separation conditions which were isocratic and gradient separation conditions. The results of system suitability test of some isocratic system elution and gradient elution system are shown in [Table 2] and [Table 3].
|Table 2: The suitability test of high-performance liquid chromatography system with isocratic elution system|
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|Table 3: The suitability test of high-performance liquid chromatography system with gradient elution system|
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The separation conditions from several different conditions can be concluded that the gradient separation conditions of 20 acetonitriles and 80 phosphate buffer of pH 3.0 are optimal conditions for performing simultaneous AML and GLI analysis processes because all conformity test parameters of the system meet the prescribed requirements [Table 4].
|Table 4: Optimum condition for suitability system test of high-performance liquid chromatography|
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Validation of the method
Validation of the analytical method was carried out using gradient elution separation conditions using acetonitrile and phosphate buffer of pH 3.0 with a ratio of 20:80; the elution conditions are shown in [Table 1]. The excitation wavelengths used for the AML and GLI separation conditions were 361 nm and 235 nm, respectively. The emission wavelengths used for AML and GLI separation conditions are 442 nm and 354 nm, respectively.
The specificity of the method is used to measure the ability of an analytical method to differentiate and measure the analyte in the presence of other components in the sample. Specificity testing was performed by injecting standard mixed solutions with a concentration of 1 μg/ml and blanks injected into HPLC. For AML, specificity testing was performed at 361 nm excitation wavelength and 442 nm emission wavelength, while for GLI, specificity testing was performed at 235 nm excitation wavelength and 354 nm emission wavelength.
Linearity testing was carried out using at least five different concentrations; the value of linearity is said to meet the requirements if the resulting correlation coefficient (r2) is >0.98. Based on the research result, the correlation coefficient of AML besylate and GLI was obtained 0.9999 and 0.9999, respectively [Table 5].
Accuracy testing was performed using three different concentrations and performed in triplicate. Accuracy was expressed by the value of %recovery; %recovery is good if it is in the range of 98%–102%. The average of %recoveries is 100.04% and 99.14% for AML and GLI, respectively. The accuracy data of the two compounds are shown in [Table 6].
Precision testing was performed using three different concentrations and performed in triplicate. Precision is expressed by the value of %relative standard deviation (RSD); %RSD is declared good if not more than 2%. The average of %RSD is 0,508% and 0,797% for AML and GLI, respectively. The precision data of the two compounds are shown in [Table 7].
Robustness testing was carried out by undergoing a change in test conditions; changes in conditions include changes in flow rate of ±0.2 ml/min and pH of the mobile phase (±0.2 units). Robustness testing was done and compared the %recovery value obtained; the %recovery value is declared to be good if it is in the range of 98%–102%. Robustness testing data from both compounds are shown in [Table 8].
| Discussion|| |
The system suitability test conducted in this experiment was done using two methods, isocratic and gradient methods. The isocratic system is a system in which the composition of the same phase of motion during measurement, while the gradient system is a system in which the mobile phase composition changes during the measurement. The variation in the comparison of the components of the mobile phase influences the peak retention time resulting from the compound and influences the polarity of the mobile phase. The effect of variation in the flow rate will affect the retention time of the compound. The system suitability of HPLC showed that the greater the flow rate, the faster the peak generated from the compound and will affect the resolution value. The resolution is smaller with an increase in the flow rate of the mobile phase. Changing the flow rate will affect the separation efficiency as it affects the widening of the peak. From the robustness test, it can be seen that the %recovery is out of acceptable range. It means that the analysis must be strictly done at the optimum pH because pH affects the ionization of AML and GLI compounds.
Validation of the method of analysis was performed before the testing process, and this study was the development of the analytical method for the AML analysis using fluorescence HPLC. The purpose of the validation of the analysis method was to confirm the inspection evidence and to have a method that in accordance with the testing objectives.
| Conclusion|| |
The optimum condition for simultaneous analysis of AML and GLI simultaneously using HPLC fluorescence without derivatization was obtained using an RP 18 (125 mm × 4 mm, i.d.) and guard column RP 18 (4 mm × 4 mm, i.d.) with mobile phase composition containing acetonitrile and phosphate buffer of pH 3.0 using a 20:80 gradient condition at flow rate 1.0 ml/min measured at λ excitation and λ emission at 361 nm and 442 nm for AML and λ excitation and λ emission at 235 nm and 354 nm for GLI. All parameters have been validated.
Financial support and sponsorship
The study was supported by the Indonesian Ministry of Research, Technology, and Higher Education (Grant-In-Aid for International Research Collaboration and Publication) for RA.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]