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Concurrent determination of Metformin and some ACE inhibitors: Its application to Pharmacokinetics
⁎Corresponding author. Tel.: +92 332 2524224. farhanchemist@gmail.com (Farhan Ahmed Siddiqui)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
This study illustrates development and validation of a simple high performance liquid chromatographic method for the simultaneous determination of metformin hydrochloride and angiotensin-converting enzyme inhibitors (captopril, lisinopril, and enalapril) in bulk dosage form and their application in pharmacokinetic studies. The quality resolute chromatogram was obtained by using a Purospher® Star RP-18 endcapped (250 × 4.6 mm id) column as stationary phase while acetonitrile-water 50:50 (v/v) as mobile phase, adjusted to pH 3.0 with phosphoric acid. Effluent was monitored at a flow rate of 1 mL min−1 at room temperature (25 °C), detector was set at 218 nm. The method was validated according to ICH guidelines. The linearity was studied over the concentration range of 10–10,000 ng mL−1 for metformin and 30–10,000 ng mL−1 for captopril, lisinopril, and enalapril, demonstrating good linearity with minimum r = 0.9964, respectively. The developed method was successfully applied to pharmacokinetic studies of metformin, lisinopril, captopril and enalapril.
Keywords
Metformin
Captopril
Enalapril
Lisinopril
ACE inhibitors and RP-HPLC
Pharmacokinetics
1 Introduction
Sufferings of people from diabetes mellitus and hypertension are most common nowadays and these diseases have been the leading cause of morbidity. Generally it has been noted that a patient suffering from diabetes also suffers from the sister disease hypertension, and the two coexists in association. Angiotensin-converting enzymes are broadly used in order to treat hypertension and heart failure. The inhibitors of the angiotensin-converting enzyme may be recommended along or in combination in combination with other drugs (Cocolas et al., 1998). Despite the fact that ACEI (angiotensin-converting enzyme inhibitors) (Fig. 1) possess contraindications, they are considered first-line therapy owing to the overwhelming benefits they bring to diabetic patients (Vaughan et al., 1997; Di Pasquale et al., 1997).
Chemical structure of all drugs.
Metformin (N,N-dimethylimidodicarbonimidic diamide) (Fig. 1), has been the crown anti-diabetic drug included in the World Health Organization Model List of Essential Medicines in the year 2008. It can be rightly said that treatment of type 2 diabetes is not complete without the administration of metformin and it is the drug of choice particularly for obese patients. Metformin helps in the reduction of LDL cholesterol and triglyceride and hence may aid weight loss (Medicines, 2007). Captopril chemically known as (2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid is an extensively prescribed antihypertensive drug and was the first inhibitor of angiotensin converting enzyme. Enalapril, an ACE inhibitor used in the treatment of hypertension and some types of chronic heart failure is chemically (2S)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-l]amino}propanoyl]pyrrolidine-2-carboxylic acid. The uniqueness of enalapril is that it was the first member of its group (ACE inhibitors) that has two attached carboxylic acid groups. Another orally active drug Lisinopril (N2-[(1S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline) belonging to ACE inhibitors is effective in lowering blood pressure, renovascular hypertension and congestive heart failure. It is a lysine analog of enalaprilate (Lancaster and Todd, 1988).
A comprehensive literature survey done in this regard revealed few analytical reports for the analysis of these drugs using HPLC alone or in combination (Europe Pharmacopoeia, 2001; Strasbourg, 2001; Amini et al., 1999; Bahmaei et al., 1997; Hillaert and Van den Bossche, 1999; Huang et al., 2006; Shimada et al., 1982; Tajerzadeh and Hamidi, 2001; Anzenbacherová et al., 2001; States Pharmacopoeia Convention, 2006; Al-Momani, 2001; Dinç et al., 2005; Wong and Charles, 1995; Sagirli and Ersoy, 2004; Rastkari et al., 2013; Sun et al., 2010, 2013; Łuczak et al., 2012; Ramusovic et al., 2012; Qin et al., 2012; Lima et al., 2009; Sun et al., 2010; Zhu et al., 2008; Rezk et al., 2013; Li et al., 2013; Sultana et al., 2011). None of these methods can be used for the simultaneous estimation of these co-prescribed and co administered drugs at the therapeutic monitoring level. There was a need to develop a simultaneous LC method for above mentioned drugs. present work describe development of a selective and sensitive HPLC methods for the determination of these drugs in pharmaceutical formulation and in human serum, using most common UV detection and having proven abilities for pharmacokinetics studies. Robustness studies were completed to ensure continuous performance of the methods in diverse analytical environments.
2 Experimental
2.1 Apparatus and materials
Metformin hydrochloride, captopril, lisinopril and enalapril reference standards were benevolently supplied by Sonaphy Aventis Limited, Efroze Chemical industries (Pvt.) Ltd., Atco Laboratories (Pvt.) Ltd. and Nabi Qasim Industries (Pvt.) Ltd., respectively. All the tablets were purchased from local market. Analytical grade phosphoric acid (85% pure), and HPLC grade methanol were purchased from Merck Marker Ltd. Water was distilled twice and was deionized by Stedec CSW-300. Ultrasonic bath (Elmasoni E 60 H), Jenway 3240 pH meter, stability Chamber (BINDER KBF 720 (E5.2)), Oven (Memmert D-91126) Schwabach FRG Germany and Sartorious TE2145 analytical balance was used in this work. Drug-free human plasma was obtained from the National Institute of Cardiovascular Disease, Karachi, Pakistan (NICVD). Caffeine was purchased from Merck.
2.2 Chromatographic condition
Shimadzu liquid chromatographic system of Shimadzu Corporation Tokyo Japan was used which consisting of Pump model (LC-20 AT VP), Detector model (SPD-20AV), Rheodyne manual injector fitted with a 20 μL loop. Chromatographic separation was obtained using Purospher® Star RP-18 end capped (25 cm × 4.6 mm id) analytical column. Class GC-10 software was used for data demand.
Acetonitrile–water 50:50 (v/v), pH was adjusted to 3.0 with phosphoric acid (85%) was used as mobile phase and acetonitrile–water 60:40 (v/v) was used as diluent. Before delivering into the system, mobile phase was filtered through 0.45 μm filter and degassed using an ultrasonic bath. Isocratic conditions were adopted using a flow rate of 1.0 mL min−1 at room temperature with detecting wave length at 218 nm. This allowed the detection of all drugs with ample sensitivity.
3 Analytical procedures
3.1 Standard preparations
Stock solution of metformin 100 μg mL−1 was prepared by dissolving 10 mg of active drug in 100 mL flask and 250 μg mL−1 of captopril, enalapril and lisinopril were prepared by dissolving 25 mg in 100 mL flask separately. Sequential dilution of these solutions were done to get a working solution with a concentration range 10–10,000 ng mL−1 for metformin and 30–10,000 ng mL−1 for captopril, enalapril and lisinopril. Diluents were used to prepare these working solutions and were scanned to get the required data for the calibration curve.
3.2 Assay procedure for pharmaceutical formulations
10 Tablets of each drug were ground in a mortar and pestle, and the required amount was carefully shifted to a 100 mL volumetric flask. The powder was dissolved in little diluent and was mechanically shaken for 5 min, then the volume was adjusted to 100 mL using the same diluent. Sequential dilutions were made to prepare solutions of different concentrations in the working range and internal standard Caffeine was added. Filtration of the samples was done using a 0.45-μm membrane filter. Linear regression equations were used to calculate the amount of metformin HCl and ACE inhibitors per tablet.
3.3 Preparation of drug serum sample
As the plasma samples were stored in the freezer at −80 °C they were allowed to thaw at room temperature before further processing. The plasma samples were centrifuged at 3000 rpm for 10 min. An aliquot of 1.0 mL was pipetted into a 10 mL polypropylene test tube and 2.0 mL of acetonitrile was added. The mixture was vortex mixed for a short while. It was then left to stand for 5 min at room temperature. The mixture was again centrifuged at 3000 rpm for 5 min. The supernatant obtained was filtered through a 0.45 μm filter. The worked out serum was then mixed in a ratio of 1:1 with drug solutions and was injected into the HPLC system.
3.4 In-vitro serum drug analysis
The availability of metformin, lisinopril, enalapril and captopril from pooled human serum was determined by the described chromatographic conditions. Blood samples of healthy volunteers were collected. Volunteers (age ranging from 22 to 28 years) were non-smokers and were not taking any other medicines. Pre written consent was obtained from all subjects. Multiple blood samples (10 mL) were collected in evacuated glass tubes through an indwelling cannula placed in the forearm veins. The blood was then slightly shaken and centrifuged at 10,000 rpm for 10 min and the plasma separated (Siddiqui et al., 2011). The obtained plasma was processed as described above and stored at −20 °C pending analysis.
3.5 In-vivo serum drug analysis
Blood samples were collected from subjects administered with drugs, in tubes containing heparin before 0.5 h and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.5, 12, 24, 30, 35, 40, 45 and 50 h intervals of administration of drug. Blood samples were centrifuged at 10,000 rpm for 10 min and plasma was separated, stored at −80 °C until use as our previous work (Siddiqui et al. 2013).
3.6 Preparation of internal standard
10 mg of caffeine was transferred to a 100 ml volumetric flask and some diluent was added to dissolve and then diluted up to the mark, further dilutions were made.
4 Results and discussion
4.1 Method optimization
The objective of this work was to develop an Rp-HPLC method for separation of these four drugs in simultaneous isocratic condition. Various hurdles were systematically countered to get to optimum conditions.
Mobile phase being the polar part of reverse phase HPLC system always has a profound effect on the separation of drug molecules which are mostly polar in nature. So the selection of mobile phase is cortically considered by chromatographers. For this purpose different ratios of methanol, acetonitrile and water were used to inspect the separation between metformin and ACEI (captopril, enalapril and lisinopril). We achieved the best separation in the mobile phase composition methanol and water 50:50 (v/v) with pH maintained at 3.1 (±1). It was observed that detector responded well at a wavelength of 218.0 nm, which was found to be the most probable isosbestic point for all studied drugs when scanned in various solvents including the diluent, thus it was selected as optimum wavelength. The peak parameters such as height, asymmetry and tailing etc were considered while maintaining flow rate, baseline drift etc. This analytical procedure does not involve the use of internal standard as there was no extraction in simultaneous determination of metformin and ACE inhibitors in human serum and in pharmaceutical dosage forms. Individual drug solutions (100 μg mL−1) were injected into the sample loop of 20 μL, elution pattern and resolution parameters were studied as a function of pH and water and organic modifiers ratios.
4.2 Method validation
4.2.1 Specificity and Robustness
This LC method under proposal is highly advantageous over the other reported method for the concurrent determination of Metformin and captopril, lisinopril and enalapril in human plasma and dosage forms. A number of other ODS (octa deca silane) columns and chromatographic systems tested confirmed negligible effect on the resolution of analytes. The best ruggedness and reproducibility was demonstrated by Purospher® Star RP-18 endcapped (25 cm × 4.6 mm id) column, hence it was adopted in the subsequent analysis. The proposed system is reasonably robust, straight forward, simple and involves commonly available UV detecting technique without any extraction fatigues. A typical reference chromatogram, Metformin and ACEIs (lisinopril, captopril, enalapril of pure standard mixture using the described condition is shown in Fig. 2. Typical chromatograms of Pharmaceutical formulation, all drugs in human serum and blank serum are shown in Fig. 3–5 respectively.
Typical chromatogram of metformin (1), internal standard (2) (caffeine), lisinopril (3), enalapril (4) and captopril (5) in reference standard.
Typical chromatogram of metformin (1), internal standard (2) (caffeine), lisinopril (3), enalapril (4) and captopril (5) in pharmaceutical formulation.
Typical chromatogram of metformin (1), internal standard (2) (caffeine), lisinopril (3), enalapril (4) and captopril (5) in human serum.
Typical chromatogram of blank human serum.
4.2.2 Linearity and range
For studying linearity standard solutions were prepared by serially diluting stock solution at five concentration level for standard reference and at seven concentration level in human serum in the range of 10–10,000 and 30–10,000 ng mL−1 for metformin and captopril, lisinopril, and enalapril, respectively which determined the linearity of the method (n = 3). Plotting the mean peak areas of metformin and captopril, lisinopril, and enalapril as a function of drug concentration gave the least-square regression calibration curves. The linearity and regression calibration curves equation are represented in Tables 1 and 2, respectively.
| Lisinopril | Enalapri | Captopril | Metformin | |
|---|---|---|---|---|
| Linearity | 30–10,000 | 30–10,000 | 30–10,000 | 10–10,000 |
| ng/ml | ng/ml | ng/ml | ng/ml | |
| Slope | 37.99 | 2.8715 | 29.22 | 69.22 |
| Intercept | 199.66 | 165.59 | 102.37 | 111.97 |
| LOD | 3.26 | 1.14 | 1.69 | 0.98 |
| LOQ | 9.26 | 6.17 | 8.22 | 2.22 |
| R | 0.9964 | 0.997 | 0.9974 | 0.9986 |
| Injected conc. ng mL−1 | Reference sample | In serum | ||||
|---|---|---|---|---|---|---|
| Recovered conc. ng mL−1 | % Recovery | %RSD n = 6 | Recovered conc. ng mL−1 | % Recovery | % RSD n = 6 | |
| Lisinopril | ||||||
| 30 | 29.66 | 98.86667 | 1.45 | 29.3 | 97.66667 | 2.24 |
| 100 | 99.41 | 99.41 | 1.17 | 98.33 | 98.33 | 1.69 |
| 500 | 501.22 | 100.244 | 1.34 | 492.5 | 98.5 | 1.77 |
| 1000 | 998.7 | 99.87 | 1.29 | 988.66 | 98.866 | 1.05 |
| 2000 | 2012.6 | 100.63 | 1.77 | 1978.5 | 98.925 | 1.35 |
| 5000 | 5044.9 | 100.898 | 1.43 | 4926.5 | 98.53 | 0.94 |
| 10,000 | 1012.4 | 10.124 | 1.69 | 9876.5 | 98.765 | 1.06 |
| Enalapril | 2.09 | |||||
| 30 | 30.11 | 100.3667 | 1.98 | 29.04 | 96.8 | 2.34 |
| 100 | 98.67 | 98.67 | 2.26 | 99.33 | 99.33 | 1.66 |
| 500 | 500.87 | 100.174 | 1.44 | 502.2 | 100.44 | 1.79 |
| 1000 | 989.4 | 98.94 | 1.69 | 993.4 | 99.34 | 1.24 |
| 2000 | 2014.5 | 100.725 | 0.88 | 1976.4 | 98.82 | 1.56 |
| 5000 | 5012.8 | 100.256 | 1.64 | 5012.8 | 100.256 | 1.38 |
| 10,000 | 9982.8 | 99.828 | 1.35 | 1027.6 | 10.276 | 1.97 |
| Captopril | ||||||
| 30 | 29.14 | 97.13333 | 2.09 | 29.14 | 97.13333 | 1.87 |
| 100 | 98.37 | 98.37 | 1.87 | 101.22 | 101.22 | 1.23 |
| 500 | 497.2 | 99.44 | 1.3 | 492.5 | 98.5 | 1.38 |
| 1000 | 988.6 | 98.86 | 1.46 | 976.5 | 97.65 | 1.45 |
| 2000 | 1994.4 | 99.72 | 1.29 | 1941.5 | 97.075 | 1.22 |
| 5000 | 4967.5 | 99.35 | 1.33 | 5032.5 | 100.65 | 1.72 |
| 10,000 | 9876.5 | 98.765 | 0.77 | 1056.8 | 10.568 | 1.69 |
| Metformin | ||||||
| 10 | 9.82 | 98.2 | 2.29 | 9.76 | 97.6 | 2.27 |
| 50 | 49.37 | 98.74 | 2.61 | 48.69 | 97.38 | 2.33 |
| 500 | 496.7 | 99.34 | 1.74 | 488.6 | 97.72 | 1.25 |
| 1000 | 992.64 | 99.264 | 1.22 | 1055.2 | 105.52 | 1.61 |
| 2000 | 1987.5 | 99.375 | 0.88 | 1974.2 | 98.71 | 1.11 |
| 5000 | 5004.5 | 100.09 | 1.09 | 5012.8 | 100.256 | 1.38 |
| 10,000 | 10121.5 | 101.215 | 1.22 | 10021.9 | 100.219 | 1.43 |
4.2.3 Accuracy and precision
The accuracy of an analytical method is defined as the similarity of the results obtained by the analytical method to the true value and precision is the degree of that similarity (Guideline, 2005; Chow et al., 2002). Repeatability and reproducibility of the method was performed as intra-day and inter-day accuracy and precision in pharmaceutical dosage forms by injecting nine replicates of three concentrations (80, 100 and 120%) into the system. Coefficient of variance (CV) and percentage recovery of each drug were also evaluated as shown in Table 3.
| Concentrations (%) | CV (n = 6) | Recovery (%) | ||
|---|---|---|---|---|
| Day 1 | Day 2 | Day 3 | ||
| Metformin | ||||
| 80 | 0.64 | 1.24 | 1.04 | 99.66 |
| 100 | 1.22 | 1.44 | 0.97 | 100.25 |
| 120 | 0.94 | 0.39 | 1.22 | 98.79 |
| Captopril | ||||
| 80 | 0.87 | 1.09 | 1.39 | 99.47 |
| 100 | 0.96 | 0.38 | 1.04 | 101.25 |
| 120 | 29 | 0.88 | 0.77 | 100.39 |
| Enalapril | ||||
| 80 | 0.66 | 0.79 | 0.79 | 100.76 |
| 100 | 1.34 | 0.52 | 0.82 | 99.87 |
| 120 | 0.47 | 0.96 | 1.06 | 100.81 |
| Lisinopril | ||||
| 80 | 0.99 | 0.63 | 0.49 | 99.38 |
| 100 | 1.23 | 0.57 | 1.22 | 101.22 |
| 120 | 0.84 | 1.06 | 1.34 | 100.38 |
4.2.4 Recovery studies
Recovery studies were performed to check the interference of formulation additives. This was done by adding a known amount of pure drug to already analyzed samples of commercial dosage forms. For percent analytical recovery values the following equation was used where Cv was the total drug concentration measured after standard addition, Cu, drug concentration in the formulation and Ca, drug concentration added to formulation. A separate analysis for all the drugs was performed to test the developed method’s precision and accuracy. All the results were found ranging from 98.2–101.6%. Results proved good recovery of the newly developed method.
4.2.5 Limit of detection (LOD) and limit of quantitation (LOQ)
The criterion for the calculation of limits of detection (LOD) and quantitation (LOQ) was the use of empirical formula i.e, 3.3(σ/s) and 10(σ/s), respectively, where σ is the standard deviation of the peak area (for six replicates) for the sample and s is the slope obtained from calibration curve equation (Green, 1996; Guideline, 1996). Using the parameters mentioned above, LOD was estimated to be 3.26, 1.14, 1.69 and 0.98 ng mL−1 while LOQ was 9.26, 6.17, 8.22 and 2.22 ng mL−1 for lisinopril, enalapril, captopril and metformin, respectively.
The uniformity of the system operation throughout the analysis was developed by initially equilibrating the column with mobile phase prior to injection of the sample into the Chromatographic system. Theoretical plates, tailing factor, resolution and repeatability were checked before starting analytical work every day. All the factors were found satisfactory and were as per (ICH, 2003) guidelines.
4.3 Applicability
4.3.1 Application to human serum (in vitro study)
The applicability of the method in human serum was checked by spiking serum sample with metformin and captopril, lisinopril, and enalapril simultaneously. The validated method was effectively applied to an in-vitro study in human plasma samples for reference and test formulations of metformin (500 mg), captopril 25 mg, lisinopril 20 mg, and enalapril 10 mg tablet formulation. Application of proposed method was conducted in the perspective of human serum, for this purpose at different concentration levels of metformin, captopril, isinopril, and enalapril were spiked with serum samples and determined in triplicate. The prepared concentrations were analogous to the intermediate concentration level of the calibration points. The sample was prepared by adding 4 mL of the drug solution (containing metformin (10 μg mL−1) and captopril, lisinopril, and enalapril (100 μg mL−1) to 4 mL of drug-free human serum. This spiked serum was made up to 10 mL with diluent and injected into the chromatograph. Further dilutions were made when and where was require. No interference was observed in the analysis as shown in Figs. 4 and 5. The obtained recoveries and %RSD are illustrated in Table 2. Statistical analysis showed that no considerable differences exist among the mean recoveries of all the drugs in serum samples.
4.3.2 Pharmacokinetic application (in-vivo study)
The method was applied to study the pharmacokinetic profile of drugs after oral administration of metformin (500 mg), captopril 25 mg, lisinopril 20 mg, and enalapril 10 mg in 24 healthy Pakistani male volunteers with a mean age of (22.5 ± 1.22 years and a mean body mass index (BMI) of 21.2 ± 2.12. Prior to analysis, the volunteers were on fast since overnight and fasted for 4 h after the dosing as well. Venous blood samples (8 ml) were collected 0.5 h before dosing and at 00.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.5, 12, 24, 30, 35, 40, 45 and 50 hrs of interval following administration.
The blood samples were treated as mentioned in experimental section. The pharmacokinetic parameters from the plasma concentration-time profile for each volunteer were evaluated. Before pharmacokinetics studies all volunteers were informed of the aim of the study, written consent was taken from all volunteers and Ethics committee approved the study protocol.
The study was performed strictly in accordance International Conference on Harmonization and USFDA (Guidance for Industry, 2001). All volunteers were checked up by general physician and found satisfactory health for conducting pharmacokinetics studies. All subjects were negative for HIV, HBSAg and HCV tests. All subjects were orally administered a single dose of test and reference drug with 240 ml of water. Drinking water was not allowed and supine position was restricted 2 h post dose. Standardized meals were provided as per requirement. The guidelines laid down by International Conference on Harmonization and USFDA (Guidance for Industry, 2001) were firmly followed in this study.
The peak plasma level of captopril was attained at almost 0.5 hrs after the administration and was found to be in good conformity with the values reported by Jankowski et al. (1995). The Plasma levels of Lisinopril achieved from healthy volunteers after the administration of a single dose was also found to be in good agreement with the value reported by Feng et al. (2012). In the same way plasma concentration time profile of enalapril and plasma concentration of metformin were also in good agreement with the reported values of Foda et al. (2010) and Mistri et al. (2007), Siddiqui et al. (2013) respectively. The mean plasma concentration-time profile of four drugs in volunteers is represented in Figs. 6–9.
Mean plasma concentration-time profile after oral administration of 20 mg of lisinopril.
Mean plasma concentration-time profile after oral administration of 10 mg of enalapril.
Mean plasma concentration-time profile after oral administration of 25 mg of captopril.
Mean plasma concentration-time profile after oral administration of 500 mg of metformin.
4.4 Stability studies
The stability studies were performed and six lots of commercially available drugs were obtained. Accelerated and long term ambient condition studies were carried out as described by ICH (2003). It was found that the commercially available lots are stable up to the six month. Two sets of studies were done one at accelerated conditions and the other one at long term ambient conditions as described by ICH (2003). Both the studies showed that all the drugs are stable in the mentioned conditions. % Recoveries of all the drugs obtained were almost 100%, Table 4.
| Month | Lisinopril | Enalapri | Captopril | Metformin | ||||
|---|---|---|---|---|---|---|---|---|
| %Recovery | RSD% | %Recovery | RSD% | %Recovery | RSD% | %Recovery | RSD% | |
| 40 °C/75% R.H | ||||||||
| 1st | 99.98 | 0.66 | 100.94 | 0.39 | 99.96 | 1.02 | 101.29 | 0.49 |
| 2nd | 99.46 | 0.94 | 100.67 | 0.89 | 99.39 | 0.86 | 100.98 | 1.34 |
| 3rd | 99.06 | 0.88 | 100.22 | 1.02 | 99.09 | 0.71 | 100.76 | 1.06 |
| 6th | 98.25 | 0.97 | 99.97 | 1.22 | 98.76 | 0.93 | 100.33 | 0.92 |
| 30 °C/65% R.H | ||||||||
| 1st | 100.56 | 0.49 | 100.39 | 0.91 | 99.41 | 0.69 | 99.69 | 0.99 |
| 2nd | 100.29 | 0.59 | 100.29 | 0.98 | 99.22 | 0.82 | 99.32 | 1.28 |
| 3rd | 100.06 | 0.38 | 100.06 | 0.52 | 99.12 | 0.86 | 99.14 | 0.58 |
| 6th | 99.89 | 0.92 | 99.76 | 0.64 | 98.85 | 0.84 | 98.69 | 1.08 |
5 Conclusion
The proposed HPLC method described in this paper provides a simple, universal, convenient and reproducible approach for the simultaneous identification and quantification of metformin, captopril, lisinopril and enalapril in biological fluids with good separation and resolution. In addition, this method has the ability to be applied to pharmacokinetic studies in human volunteers and Analytical results are accurate and precise with good recovery. This assay method was also successfully applied in raw materials and pharmaceutical formulations. Linearity, accuracy, precision, limit of detection and quantification, specificity were established. Hence this method is beneficial for quality control laboratories as well as bio-analytical laboratories. This method can not only be used to study dissolution and pharmacokinetics but also therapeutic monitoring of the mentioned drugs can be studied in diabetic and hypertension patients having simultaneous drug prescription.
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