1. Introduction
Worldwide, at least one in every four people suffer from metabolic syndrome (MetS) [
1]. Obesity is a crucial factor in getting MetS, and this condition is reaching epidemic proportions. Only in the U.S., 68% of the population is considered overweight or obese [
2]. MetS is defined as a constellation of interrelated risk factors that appear to promote diabetes and cardiovascular diseases [
3]. Commonly, a patient with MetS will suffer from hypertension, hypercholesterolemia, and diabetes simultaneously [
4]. For this reason, the World Health Organization’s Global Hearts has recommended the use of combination therapy (multiple drugs for the treatment of two or three different illnesses at the same time) for improving the treatment of MetS [
5]. The development of novel pharmaceutical formulations not only demands the finding of the optimal combination of active ingredients, but it may also require specific analytical methodologies suitable for determination of the multiple drugs that are present in the formulation.
Some of the most used Active Pharmaceutical Ingredients (API) in the development of formulations for combination therapy for the treatment of metabolic syndrome are: bezafibrate (BZT, pK
a = 3.6), a representative fibrate widely used in the treatment of hypercholesterolemia [
6,
7]; gliclazide (GZD, pK
a = 5.8), an oral hypoglycemic agent, belonging to second-generation sulphonylureas, which is used in type II diabetes (non-insulin-dependent diabetes mellitus) [
8,
9,
10]; glimepiride (GMP, pK
a = 6.2), an oral blood-glucose-lowering drug of the third-generation sulfonylureas, used for the treatment of diabetes [
11,
12,
13]; telmisartan (TEL, pK
a = 4.5), a synthetic analog of angiotensin II receptor blocker used in the management of hypertension [
14,
15,
16]; and carvedilol (CVD, pK
a = 7.5), a non-selective beta-alpha blocker 1, used in the treatment of high blood pressure [
17,
18,
19]. Since all these compounds contain aromatic rings in their structure (see
Figure 1), they cannot be simultaneously quantified following a UV−Vis spectrophotometric analytical method since all of them absorb in the same region of the UV-spectrum. Therefore, separation techniques such as high-performance liquid chromatography with a diode-array detector (HPLC-DAD) are required.
Different HPLC chromatographic methods have been reported for individual determination of BZT [
20,
21,
22], GZD [
8,
9,
23], GMP [
11,
12,
24], TEL [
14,
15,
25], and CVD [
17,
18]. However, fast methodologies for the quantification of more than two drugs in a single run are limited. The authors of these studies only focus their analytical methods on drugs prescribed for the treatment of a single type of disease, either hypercholesterolemia [
26,
27,
28,
29], hypertension [
19,
30,
31,
32] or diabetes [
33,
34,
35,
36].
There are only two analytical methods reported where authors quantify APIs for the three main illnesses of MetS [
7,
37]. However, these have the disadvantage of long retention times and high limits of detection and quantification. Analytical methods that allow fast simultaneous quantification of five or more drugs may facilitate quality assurance, quality control processes in clinical trials for combined therapy. A short analysis time is a significant parameter to consider since it reduces the number of toxic organic solvents. Economizing in using solvents and supplies and energy consumption would reduce the environmental impact providing the pharmaceutical industry with efficient greener analytical methodologies for developing formulations, quality assurance, quality control, and potential clinical studies [
38].
In the present study, a rapid HPLC-DAD methodology was developed for simultaneous quantification of bezafibrate (hypercholesterolemia), gliclazide and glimepiride (diabetes type II), carvedilol and telmisartan (hypertension) in bulk and applied to the quantification of pharmaceutical commercial tablets. The analytical method presented was validated by International Conference on Harmonization guidelines. There is no previous published analytical method in which these five APIs can be determined simultaneously in a single chromatographic run to the authors’ best knowledge.
2. Materials and Methods
2.1. Materials and Reagents
All active pharmaceutical ingredients: CVD (PHR1265), TEL (PHR1855), BZT (72516), GZD (G2167), and GMP (PHR1617) with purities higher than 99% were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. Methanol (HPLC grade), acetonitrile (ACN, HPLC grade), orthophosphoric acid (analytical reagent grade) and potassium dihydrogen phosphate anhydrous (analytical reagent grade) were purchased from J.T. Baker Inc. (Columbus, OH, USA). Milli Q (Millipore, Milford, MA, USA) grade water was used for the preparation of buffer for the HPLC mobile phase and all aqueous solutions.
2.2. Equipments
High-performance liquid chromatography (HPLC) analysis was performed with an Agilent 1200 Series HPLC system (Agilent Technologies, Waldbronn, Germany) consisting of a quaternary pump (G1311A quat pump), degasser (G1322A Degasser), DAD SL diode-array detector (G1315D DAD), thermostatted column compartment (G1316A COLCOM), and autosampler thermostat (G1330B FC/ALS Therm). A Hypersil GOLD C18 Selectivity, 5 µm (150 × 4.60 mm2) column, with a precolumn guard cartridge, was used (Thermo scientific, Bellefonte, PA, USA). Analysis of chromatographic peaks and calculation of the areas were performed using the ChemStation for LC 3D systems software (Agilent Technologies, Waldbronn, Germany). A C18 column was selected because it is an appropriate stationary phase to achieve the separation of acidic and neutral drugs, providing an excellent choice for the separation of these drugs at short retention times
2.3. Chromatographic Conditions
The simultaneous analysis of the five drugs was carried out with a mobile phase of acetonitrile: phosphate buffer in the ratio of 50:50 v/v with a pH 3 (adjusted with orthophosphoric acid). ACN was selected because of its low UV cutoff and lower viscosity in mixtures with water (compared to methanol). Multiple water/acetonitrile ratios were tested to find that coelution was avoided in the 50/50 ratio. Finally, a pH = 3 for the mobile phase was selected because it fell below the analytes’ pKa values, ensuring that these existed in the unionized form.
The mobile phase was filtered through a 0.2 µm pore size nylon membrane of 47 mm diameter (Thermo scientific, Dreieich, Germany). The isocratic flow rate of the mobile phase was 1 mL/min, column temperature was set to 25 °C, and the injection volume was 20 µL. The selected absorption wavelengths for detection were 242 nm for CVD, 298 nm for TEL, and 230 nm for BZT, GZD and GMP.
2.4. Preparation of Standard Solutions
Individual standard solutions were prepared for the construction of calibration curves for each drug. A 10 mg dose of API was added to 50 mL of methanol and stirred until complete dissolution. The solution was then transferred into a 100 mL volumetric flask, and water was added to complete the volume (stock solutions of 100 μg mL−1). A series of standard solutions was prepared using the appropriate dilution of stock solution in water−methanol (50/50) to reach eight concentrations: 0.5, 2.5, 5, 7, 10, 15, 20, and 25 μg mL−1. These standard solutions were used for the determination of linearity and the construction of calibration curves.
2.5. Working Standard Solution
A mixture of CVD, TEL, BZT, GZD, and GMP was prepared using 10 mL from each stock solution into a 100 mL volumetric flask. Water−methanol (50/50) was added to complete the volume, obtaining a working standard solution with 10 μg mL−1 of each API. Working standard solutions were analyzed to evaluate the feasibility of chromatographic separation of all drugs for their simultaneous determination. The area of every peak observed, corresponding to each API, was compared with the area of standard solution, and methodology was accepted if relative error RE% was <15%.
2.6. Analytical Method Validation
The validation of the method was carried out evaluating solution stability, linearity range, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision, according to International Conference on Harmonization (ICH) validation guidelines [
39].
2.6.1. Solution Stability
A common practice in industry is the use of an autosampler for continuous automatized analysis; thus, it is important to evaluate the API’s standard solution stability over several hours since some drugs may undergo degradation [
30]. Therefore, to determine the stability of CVD, TEL, BZT, GZD and GMP dilutions from stock standard solutions (10 μg mL
−1) were assayed after 72 h of storage at −20, 4, and 25 °C. For accelerated studies, dilutions from the five APIs’ stock solutions were also stored for 8 h at 80 °C. The area corresponding to the peak of API was compared against the area obtained for fresh standard solutions. Three replicates were performed, and stability of API was accepted if percentage coefficient of variation (CV%) was < 3% and percentage relative error (%RE) was <15% for the areas.
2.6.2. Linearity
The linear range analysis for each API was carried out analyzing standard solution at five concentrations: 0.5, 2.5, 5, 7, 10, 15, 20, and 25 μg mL
−1, prepared from a stock solution. Every solution was evaluated in five replicates. The corresponding area of the peak was recorded and plotted as a function of concentrations. The range was considered linear if the correlation coefficient (R
2) was larger than 0.999 [
29,
39].
2.6.3. Limit of Detection and Quantification
LOD and LOQ were determined based on the standard deviation of the response and the slope, according to ICH. LOD was calculated according to equation 1 and, LOQ was calculated according to Equation (2) [
39].
where σ corresponds to standard deviation of the
y-intercepts of regression line and S is the slope of calibration curve. Both LOD and LOQ were validated by independent analysis of three different concentrations, at LOD and LOQ, under and above.
2.6.4. Precision and Accuracy
To determine the method’s precision and accuracy, three independent concentrations (2.5, 10 and 25 μg mL
−1) were measured for each API. Each concentration was evaluated as five replicates under two different conditions: (1) all solutions were evaluated the same day (repeatability intraday), and (2) samples were evaluated on three different days (repeatability interday). Precision was determined by repeatability intraday and interday, and this parameter was expressed as CV%. A precision (CV%) less than or equal to 15% is acceptable. In the case of accuracy, this parameter is expressed by relative error (RE%), and a value less than or equal to 15% is acceptable [
40,
41].
2.7. Sample Solutions of Commercially Available Drug Products
In order to apply the methodology to commercially available products, tablets of carvedilol (25 mg), telmisartan (40 mg), bezafibrate (200 mg), gliclazide (60 mg), and glimepiride (2 mg) were quantified following two different procedures: (1) five tablets of each commercial product were weighed and ground with mortar and pestle for 5 min. After the tablets were ground, an amount equivalent to 10 mg of API was used to prepare sample solutions in 100 mL water−methanol 50/50 (100 μg mL−1). The final concentration of solutions was 10 μg mL−1; this concentration was the commercial API’s nominal concentration. (2) The content of a single tablet was quantified; the tablet was weighed and ground with a mortar and pestle for 5 min and then dissolved in 50/50 water−methanol.
Samples solutions of commercially available drug products were analyzed individually, and the concentration obtained was compared with the nominal concentration of 10 μg mL−1. The API’s quantified mass in a single tablet was also compared to the labeled product’s expected content. Recovery percentages and relative errors were reported. Sample solutions were prepared and analyzed in triplicate.
2.8. Working Sample Solutions of Commercially Available Drug Products
The simultaneous quantification of the five commercially available drug products was evaluated. Working sample solutions were prepared by addition of 10 mL from each sample solution (100 μg mL−1) into 100 mL volumetric flask and water−methanol (50/50) to complete volume, obtaining a working sample solution with 10 μg mL−1 of each commercial API.
4. Conclusions
A novel isocratic reverse-phase HPLC-DAD methodology was developed for the simultaneous quantification of bezafibrate, gliclazide, glimepiride, telmisartan, and carvedilol. The method developed was rapid, linear, reproducible, and a greener analytical method. The results showed that the limit of detection and limit of quantification for the simultaneous determination of bezafibrate, gliclazide, glimepiride, and telmisartan were improved compared to previously reported methodologies. These results contribute to a methodology that could be applied in quality control in the pharmaceutical industry or the development of pharmaceutical formulations or clinical studies.