A green HPLC method for the determination of apixaban in pharmaceutical products: Development and validation
-
Erten Akbel
,
İbrahim Bulduk
Abstract
Apixaban (APX) is a novel anticoagulant drug used in the treatment of ischemic stroke and venous thromboembolism. In this study, two different chromatographic methods were developed for the determination of APX in pharmaceutical products. In the first method, an Agilent C18 column (250 mm × 4.6 mm, 5 μm) was used, and the temperature was kept constant at 30°C. The mobile phase was chosen to be 0.1% trifluoroacetic acid solution and acetonitrile (65:35, v/v), and isocratic elution was applied. The flow rate of the mobile phase was found to be 1.0 mL·min−1 and the injection volume was 20 µL. The detection was carried out at a wavelength of 276 nm using a UV detector. In the second method, ethanol was used as an organic modifier. The only difference between these methods was the organic modifier. All other conditions of the methods were the same. Both chromatographic methods were validated in accordance with ICH guidelines for various parameters such as selectivity, linearity, accuracy, precision, detection and quantification limit, and robustness. The determination coefficients of chromatographic methods were greater than 0.999 in the concentration range of 5–30 mg·mL−1 of APX. Later, these chromatographic methods were applied to tablet formulations. Comparison of the obtained results in terms of mean was made using Student’s (t) test, and comparisons in terms of standard deviations were made using the Fisher (F) test. It was observed that there was no significant difference between these methods. These two methods were then evaluated using AGREE-Analytical greenness metric software. The chromatographic method using ethanol as an organic modifier has been proposed as an excellent eco-friendly and analyst-friendly alternative for the determination of APX in pharmaceutical formulations.
Graphical abstract
1 Introduction
High-performance liquid chromatography (HPLC) is one of the most widely used technologies in pharmaceutical analysis. HPLC is widely used in quality control of pharmaceutical formulations, product stability testing, determination of drug degradation products, characterization of drug impurities, and analysis of biological materials [1]. In pharmaceutical laboratories, HPLC methods are usually developed based on the reverse-phase mode using a hydrophobic stationary phase and a polar mobile phase, and an ultraviolet/visible (UV/Vis) detector mode. Therefore, the compatibility of the mobile phase with the detector is often considered when developing a pharmaceutical analysis technique. Mobile phases used in HPLC typically consist of a combination of water (with additions to alter the pH and ionic strength) and organic modifiers such as acetonitrile/methanol [2]. Acetonitrile and methanol are often used and preferred in HPLC analysis due to their unique chromatographic properties such as complete miscibility with water, the relatively low viscosity of their aqueous solutions, low UV cut-off wavelength, low chemical reactivity with most sample types and HPLC, and high purity availability [2,3].
Despite these unique chromatographic properties, acetonitrile and methanol pose some problems in terms of health safety and ecological impact. Acetonitrile is a volatile, flammable, and toxic chemical. Although methanol is biodegradable and less toxic than acetonitrile, it is classified as a hazardous solvent due to its toxicity and the difficulty of waste disposal [4,5]. The amount of waste produced during HPLC analysis can never be underestimated. A conventional HPLC device produces about 1–1.5 L of waste per day, which corresponds to about 500 L per year [3].
Although this amount is very small compared to the amount of waste produced by large industrial production companies, hundreds of liquid chromatography devices are used in the quality control and research and development laboratories of large pharmaceutical companies, and tons of toxic waste are generated every day. In addition, due to technological developments, the use of HPLC is becoming increasingly widespread, and at the same time, the amount of waste is increasing. These wastes, which contain a large proportion of acetonitrile and methanol, should be disposed of as chemical waste. This situation increases the burden of environmental waste disposal of laboratories and imposes quite high costs. The issue of developing environmentally friendly HPLC methods is of great interest among analytical chemists who are looking for new alternatives to replace polluting and toxic chemicals with less polluting ones [6].
All HPLC methods have the potential to be more environmentally friendly and user-friendly at all stages of analysis, from sample preparation to final determination [7,8,9,10]. The mobile phases used in HPLC are classically a mixture of water (containing additives to adjust the pH and ionic strength) and organic solvents. HPLC operators usually use acetonitrile and methanol as organic modifiers. However, both of these are classified as hazardous chemicals due to their toxic effects and the great importance given to the safe detoxification of waste. For this reason, they should not be preferred as much as possible [5].
Since it is quite difficult to develop an HPLC method without the use of organic solvents, it is necessary to replace acetonitrile and methanol with other less toxic organic solvents in order to make this method more environmentally friendly and minimize its negative effects on the operator's health [11]. Ethanol is one of the organic solvents that are environmentally friendly [12]. Ethanol is less harmful from the point of view of the operator and environmental health compared to acetonitrile and methanol. The fact that ethanol has a low vapor pressure causes less ethanol to evaporate and the operator to breathe more. Ethanol is also widely available and is cheaper than other organic solvents. Especially in developing countries, laboratories with scarce resources can easily obtain ethanol [3]. In addition, ethanol has cheaper disposal costs compared to other organic solvents. This is a big advantage for cities where chemical waste disposal is expensive. It has similar properties to acetonitrile and methanol in terms of chromatography [13]. Column-filling materials and adsorption mechanisms are quite similar. It has similar separation mechanisms when different solvents are used. When ethanol is used instead of acetonitrile or methanol, similar peak yields are obtained in the chromatographic separation of mixtures [14]. According to the organic solvent classification, ethanol is in the same group as methanol in terms of selectivity [15]. However, there are two main disadvantages to using ethanol in HPLC [16]. The first of these is that ethanol has a higher UV cutting value (210 nm) than methanol and acetonitrile. This can lead to an increase in background noise and a significant shift in the baseline, resulting in a decrease in sensitivity when using the gradient elution system [2]. The second disadvantage is that the viscosity of the ethanol/water mixture under laboratory conditions is higher than methanol/water and acetonitrile/water mixtures. High viscosity can cause high column pressure [17]. Column temperature affects selectivity, yield, and mobile phase viscosity. Slightly increasing the column temperature reduces the viscosity of the mobile phase, resulting in a decrease in the column pressure.
Apixaban (APX) has the chemical name (1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide). The chemical structure of APX is shown in Figure 1. APX is a novel anticoagulant drug used in the treatment of ischemic stroke and venous thromboembolism [18,19,20].
The chemical structure of APX.
Analytical procedures previously published in the literature were scanned to determine the amount of APX in pharmaceutical products and biological matrices. Spectrophotometric [21,22,23,24], spectrofluorometric [25], electrochemical [26], HPLC [27,28,29,30,31,32,33], high-performance thin-layer chromatographic [34], and liquid chromatographic coupled with tandem mass spectrometry [35,36,37,38,39] methods were reported for the assay of APX.
Most of these methods are quite complex and require expensive equipment, toxic organic solvents, and special chemicals. In the literature review, only one green high-performance thin-layer chromatography (HPTLC) method was reported for the determination of APX in pharmaceutical tablets [34]. The green HPLC method was not found. Therefore, the aim of this study was to develop and validate an environmentally and analyst-friendly liquid chromatography method in which ethanol is used as a mobile-phase organic modifier for APX quantification in pharmaceutical products by a simple extraction procedure.
2 Materials and methods
2.1 Instruments
An Agilent 1260 series HPLC system (Wilmington, DE, USA) was used for the HPLC analysis. This chromatographic system is equipped with a degasser, quaternary pump, autoinjector, and ultraviolet detector, and the column oven temperature was 30°C. Chemstation software and Extend C18 (5 μm, 250 mm × 4.6 mm) column (Agilent, USA) were used for analysis. A Mettler Toledo pH meter equipped with a glass electrode was used for pH measurement. Millipore Milli-Q water purification system (Milford, MA, USA) was used for the preparation of ultrapure water.
2.2 Reagents
The APX United States Pharmacopeia Reference Standard, ethanol (≥99.9%), acetonitrile (≥99.9%), and trifluoroacetic acid (≥99.0%) were purchased from Sigma-Aldrich Chemie GmbH (Istanbul, Turkey). All of the other compounds were of analytical grade. APX tablets (Eliquis, 5 mg, Batch number: DE7421) used in this study were purchased from a local pharmacy (Afyonkarahisar, Turkey). Ultrapure water (conductivity <0.05 µS·cm−1) was produced using a Milli-Q water purification system and used in experimental studies.
2.3 APX standard solutions
About 25 mg of APX reference standard was weighed precisely and transferred into a 50 mL of volumetric flask. It was dissolved in an ultrasonic bath by adding 20 mL of ethanol until a clear solution was produced, balanced to room temperature (25°C), and the volume was completed to 50 mL with ultrapure water. From this stock standard solution, standard solutions were prepared by diluting with ultrapure water so that the final concentrations were 5, 10, 15, 20, 25, and 30 µg·mL−1.
2.4 APX sample solution
Ten APX-containing tablets were precisely weighed. It was ground into a fine powder in a dry and clean mortar. The tablet powder equivalent to 25 mg of APX was then transferred into a 50 mL volumetric flask. About 20 mL of ethanol was added and shaken in a rotary shaker for 20 min to ensure complete dissolution. The volume was completed with ultrapure water. The mixture was sonicated for 10 min and then filtered through a 0.45 mm membrane filter. The sample solution was diluted with ultrapure water from the prepared stock sample solution and prepared at a concentration of 20 µg·mL−1.
2.5 Determination of λ max for APX
Standard solutions in the concentration range of 5–30 µg·mL−1 were scanned in a UV spectrophotometer (Shimadzu UV-1800 spectrophotometer) device at a wavelength range of 200–400 nm. The overlapping spectrum of standard solutions is shown in Figure 2.
Overlaid spectra of standard solutions in the concentration range of 5–30 µg·mL−1.
2.6 Validation parameters
Chromatographic methods have been validated in accordance with the International Conference on Harmonization recommendations on the validity of analytical procedures [40,41]. Validation parameters such as linearity, specificity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness were investigated. Based on these considerations, a specific concentration range (5–30 µg·mL−1) was chosen for the validation procedure.
2.7 Selectivity
Standard, sample, and mobile phase solutions were injected into the chromatographic system to evaluate the selectivity of the chromatographic methods. Chromatograms were compared and it was examined whether there were interference peaks in the retention time region of the APX peak.
2.8 Linearity
The linearity of the chromatographic methods was evaluated by injecting six standard APX solutions into the HPLC system in the concentration range of 5–30 µg·mL−1. Three replicate analyses were performed on three different days. A calibration curve was created by plotting the analyte concentration versus the peak area. The slope and intercept of the regression equation were calculated using linear regression analysis based on the least-squares method. The linearity of the method was measured by the absolute mean recovery, relative standard deviation (RSD), and R 2 of the resulting calibration curve.
2.9 System suitability
A standard solution of 20 µg·mL−1 was injected into the chromatographic system six times to determine the suitability of the system. The peak area, retention time, tailing factor, and theoretical plate number values were recorded from chromatograms. The RSD% values of peak areas and retention times were calculated for six injections.
2.10 Accuracy
The accuracy of the analytical methods was determined by spiking three different amounts of APX standard into the sample solution. The standard was added to the sample solution (20 mg·mL−1) at 80%, 100%, and 120% of the APX content. The solutions obtained were injected into the chromatographic system. The % recovery values of the added standard amount were calculated. Triplicate tests were performed for each concentration.
2.11 Precision
The precision of the chromatographic methods was evaluated based on the intra-day and inter-day reproducibility of the method. Intra-day reproducibility was evaluated by determining the RSD of the peak areas obtained from three injections of the standard solution (20 μg·mL−1) on the same day. For inter-day reproducibility, the same standard solution was injected three times on three consecutive days. The RSD of the obtained areas was determined and evaluated.
2.12 LOD and LOQ
The detection limit was calculated by the formula LOD = 3.3σ/S using the standard deviation (SD) of the calibration curve in the developed chromatographic methods. The quantification limit was calculated by the formula LOQ = 10σ/S using the SD of the calibration curve in the developed chromatographic methods. In these formulas, σ is the SD of the point where the calibration curve intersects the y-axis and S is the slope of the calibration curve.
2.13 Robustness
Minor deliberate changes to method conditions were made to assess the robustness of the chromatographic methods. Minor changes were performed in the flow rate of the mobile phase (±0.1 mL·min−1), the organic solvent content in the mobile phase (±2%), the column temperature (±5°C), and the pH value of the mobile phase (±0.05), and the effect of these changes on the system suitability parameters were observed. These effects were investigated by triplicate analysis of the standard solution at a concentration of 20 µg·mL−1.
2.14 Solution stability
The stability of the standard solution was assessed by storing it at different conditions including 25°C (ambient conditions) for 12, 24, 36, and 48 h, and 4°C (refrigerator temperature) for 10 days. At the end of each storage time, the solutions were injected into the HPLC system and the results were compared with that of freshly prepared sample solutions. The stability analysis was performed using an analytical solution at a concentration of 20 μg·mL−1.
2.15 Evaluation of greenness of chromatographic methods
The proposed chromatographic methods were evaluated using the AGREE-Analytical greenness metric software. AGREE is a metric system for evaluating the environmentalism of analytical procedures based on the principles of significance. AGREE is an easy-to-apply program with user-friendly software, which has been expanded by incorporating 12 basic principles in greenery assessment, allowing flexible working by allowing weight assignment, and an easily interpretable color pictogram output showing strong and weak points. The analytical greenness score is the weighted average of the benchmark scores. It is shown in the middle of the graph, rounded to two decimals, and its value ranges from 0.0 (lowest score) to 1.0 (excellent score). The chart is a visual representation of the score itself, criterion scores, and criterion weights [42,43].
3 Results
3.1 Method development
All conditions were optimized to develop an efficient chromatographic method for the quantification of APX in pharmaceutical preparations. First, to determine the wavelength at which APX absorbs UV rays at a maximum level, standard solutions in the concentration range of 5–30 µg·mL−1 were scanned with a spectrophotometer device against ultrapure water in the wavelength range of 200–400 nm. When the spectra were examined, it was observed that APX at 276 nm wavelength absorbs UV rays maximally. Also, baseline noise was minimal at this wavelength. It has been observed that there is no interference from drug additives or filler materials at this wavelength in pharmaceuticals. The overlapping spectrum of standard solutions is shown in Figure 2.
Chromatographic conditions were optimized to obtain good peak parameters such as a good peak shape, a good tailing factor, a short retention time, and a high theoretical plate count. At the beginning of the study, mobile phases consisting of many buffer systems were investigated but the required system compatibility properties could not be achieved. Different types of columns and different lengths were tested. However, the system suitability parameters were found to be weak. Good peak parameters were obtained with an Agilent C18 (250 mm × 4.6 mm, 5 µm) column. Different ratios of water/methanol, water/acetonitrile, and methanol/acetonitrile mixtures were tested as the mobile phase. Acetonitrile and ultrapure water (20:80, v/v) were initially used as the mobile phase, resulting in a very long analysis time. Then, the water component of the mobile phase was acidified with trifluoroacetic acid (pH 2.0). Under these conditions, the sample solution was injected to detect both impurities that could interfere with the APX peak and the presence of drug matrix components that could remain in the column longer under the specified conditions. In addition, sample solutions were injected sequentially into the system with a 10 min analysis period and it was observed that no impurities were transferred from one analysis to the next. Therefore, the analysis time was determined as 10 min. In addition, the column temperature was chosen as 25°C due to its cost-effectiveness as well as many advantages such as high column efficiency, low column pressure, and a suitable peak shape.
As a result, the chromatographic method was optimized as follows: an Agilent C18 column (250 mm × 4.6 mm, 5 µm) was used and the temperature was kept constant at 25°C. The mobile phase was chosen as trifluoroacetic acid solution (0.1%) and acetonitrile (65:35, v/v), and isocratic elution was applied. The flow rate of the mobile phase was found to be 1.0 mL·min−1 and the injection volume was 20 µL. Detection was carried out using a UV detector at a wavelength of 276 nm.
3.2 Selectivity
Standard, sample, and mobile phase solutions were injected into the chromatographic system to evaluate the selectivity of the chromatographic methods. The three chromatograms were compared and examined for interfering peak(s) around the analyte peak. No peak interfering with the APX retention time was observed in either method. Chromatograms of the standard, sample, and mobile-phase solutions produced by Method II are shown in Figure 3.
Chromatogram of (a) the standard solution (25 µg·mL−1), (b) the sample solution (20 µg·mL−1), and (c) the mobile phase.
3.3 Linearity
The standard solutions (5, 10, 15, 20, 25, and 30 µg·mL−1) were prepared in three replicates from the stock standard solution (500 µg·mL−1) by diluting with ultrapure water. These standard solutions were injected into the chromatographic system, and the peak areas and retention times of the analyte were recorded. Average peak areas were calculated for each concentration level. A calibration graph was drawn with the peak area values versus the concentration of the standard solution versus the peak area. Linearity data of chromatographic methods were evaluated by regression analysis. The regression equation, slope, and intercept were calculated using linear regression analysis based on the least-squares method. The linearity of the method was measured by the absolute mean recovery, RSD, and R 2 of the resulting calibration curve. All linearity data are given in Table 1. The overlaid chromatograms of APX standard solutions for Methods I and II are presented in Figure 4.
Regression analysis results of chromatographic methods
| Parameter | Method I (acetonitrile) | Method II (ethanol) |
|---|---|---|
| Concentration range (μg·mL−1, n = 6) | 5–30 | 5–30 |
| Slope of the regression equation | 37.497 | 37.735 |
| Intercept of the regression equation | 9.3738 | 4.6717 |
| Determination coefficient | 0.9999 | 0.9998 |
| Retention time (min) | 5.34 | 4.51 |
| Detection limit (μg·mL−1) | 0.50 | 0.70 |
| Quantification limit (μg·mL−1) | 1.60 | 2.00 |
| Recovery (%), n = 3 | 99.59–100.43 | 99.64–100.87 |
(a) The overlaid chromatograms of standard solutions. Method I: column, Agilent C18 (250 mm × 4.6 mm, 5 µm); mobile phase, trifluoroacetic acid solution (0.1%) and acetonitrile (65:35, v/v); flow rate, 1.0 mL·min−1; injection volume, 20 µL; detection; 276 nm; and temperature, 25°C. (b) The overlaid chromatograms of standard solutions. Method II: column, Agilent C18 (250 mm × 4.6 mm, 5 µm); mobile phase, trifluoroacetic acid solution (0.1%) and ethanol (65:35, v/v); flow rate, 1.0 mL·min−1; injection volume, 20 µL; detection, 276 nm; and temperature, 25°C.
3.4 Accuracy
The accuracy of the chromatographic methods was determined by spiking three different amounts of APX standard into the sample solution. The standard was added to the sample solution (20 mg·mL−1) at 80%, 100%, and 120% of the APX content. The solutions obtained were injected into the chromatographic system. The % recovery values of the added standard amount were calculated. Triplicate tests were performed for each concentration. The recovery percentages ranged between 99.67% and 99.72% in Method I and between 99.55% and 99.63% in Method II. It was observed that the RSD values were a maximum of 0.2187 in Method I and a maximum of 0.3887 in Method II. The results of the recovery studies are presented in Table 2.
Accuracy data of chromatographic methods
| Method | Amount added (μg·mL−1) | Average recovery (%) | SD | RSD (%) |
|---|---|---|---|---|
| Method I | 16 | 99.67 | 0.218 | 0.2187 |
| 20 | 99.69 | 0.125 | 0.1254 | |
| 24 | 99.72 | 0.103 | 0.1033 | |
| Method II | 16 | 99.55 | 0.387 | 0.3887 |
| 20 | 99.59 | 0.179 | 0.1797 | |
| 24 | 99.63 | 0.124 | 0.1244 |
3.5 Precision
Intra-day precision was assessed by determining the RSD values of the retention times and the areas of peaks obtained from three injections of the standard solution (20 μg·mL−1) on the same day. The RSD values of the peak areas and retention times were found to be below 1.00% in chromatographic methods. The intra-day precision results are given in Table 3. For inter-day precision, the same standard solution was injected three times a day for three consecutive days. The relative standard deviations of the retention times and the areas of the obtained peaks were determined and evaluated. The RSD values of the retention times and peak areas of APX peaks were found to be below 1.00% in chromatographic methods. The inter-day precision results are given in Table 4. Our data show that the methods are suitable for validation requirements.
Intra-day precision results of chromatographic methods
| Sample no. | Method I | Method II | ||||
|---|---|---|---|---|---|---|
| Retention time (min) | Peak area | Assay (%) | Retention time (min) | Peak area | Assay (%) | |
| 1 | 5.359 | 763.03 | 100.07 | 4.509 | 756.36 | 99.97 |
| 2 | 5.367 | 762.56 | 100.01 | 4.512 | 757.40 | 100.11 |
| 3 | 5.363 | 761.92 | 99.92 | 4.513 | 755.93 | 99.92 |
| Mean | 5.363 | 762.50 | 100.00 | 4.511 | 756.56 | 100.00 |
| SD | 0.004 | 0.5572 | 0.073 | 0.002 | 0.7565 | 0.100 |
| RSD (%) | 0.075 | 0.0731 | 0.073 | 0.046 | 0.1000 | 0.100 |
Inter-day precision results of chromatographic methods
| Day | Injection no. | Method I | Method II | ||||
|---|---|---|---|---|---|---|---|
| Retention time (min) | Peak area | Assay (%) | Retention time (min) | Peak area | Assay (%) | ||
| First | 1 | 5.367 | 761.53 | 99.80 | 4.513 | 756.36 | 99.99 |
| 2 | 5.363 | 762.86 | 99.97 | 4.512 | 755.40 | 99.86 | |
| 3 | 5.370 | 761.02 | 99.73 | 4.509 | 754.93 | 99.80 | |
| Second | 4 | 5.370 | 763.81 | 100.10 | 4.508 | 756.31 | 99.98 |
| 5 | 5.368 | 762.32 | 99.90 | 4.513 | 756.92 | 100.06 | |
| 6 | 5.362 | 763.17 | 100.01 | 4.509 | 756.24 | 99.97 | |
| Third | 7 | 5.363 | 764.22 | 100.15 | 4.510 | 757.47 | 100.14 |
| 8 | 5.370 | 764.41 | 100.18 | 4.508 | 756.57 | 100.02 | |
| 9 | 5.364 | 764.33 | 100.16 | 4.509 | 757.83 | 100.18 | |
| Mean | 5.366 | 5.366 | 763.07 | 100.00 | 4.510 | 756.45 | |
| SD | 0.003 | 0.003 | 1.2472 | 0.163 | 0.002 | 0.9128 | |
| RSD (%) | 0.063 | 0.063 | 0.1634 | 0.163 | 0.045 | 0.1207 | |
3.6 System suitability
To evaluate the suitability of the system, the primary parameters were determined for a standard solution at a concentration of 20 µg·mL−1. The values of system suitability parameters are presented in Table 5. The peak symmetry of APX was perfect. In addition, the variability of peak areas and retention times were quite low. The correlation coefficients of the calibration curve in chromatographic methods are above 0.999, indicating that the methods are suitable for samples with simple or complex matrices.
Results of system suitability tests for chromatographic methods (n = 6)
| System suitability parameter | Method I | Method II |
|---|---|---|
| Asymmetry factor | 0.8580 | 0.7346 |
| RSD (% of peak areas) | 0.1634 | 0.1207 |
| RSD (% of retention times) | 0.0630 | 0.0450 |
| Tailing factor | 1.1471 | 1.3052 |
| Theoretical plate count | 6,391 | 5,651 |
3.7 Robustness
The results of the robustness study have shown that the linearity, accuracy, and recovery of chromatographic methods are not affected by small changes in the critical method parameters such as the column temperature, flow rate, organic solvent content, and pH value of the mobile phase. The results related to the robustness study are presented in Table 6. The average recovery for all tests was between 99.71% and 100.45%, and the RSD% level was less than 0.82.
Results of robustness tests
| Method | Parameters | Values | Average recovery (%) | RSD (%) |
|---|---|---|---|---|
| Method I | Flow rate of the mobile phase | 0.90 mL·min−1 | 100.45 | 0.22 |
| 1.10 mL·min−1 | 99.79 | 0.15 | ||
| Column temperature | 20°C | 99.98 | 0.17 | |
| 30°C | 100.13 | 0.27 | ||
| Acetonitrile content of the mobile phase | 33% | 99.95 | 0.79 | |
| 37% | 100.41 | 0.53 | ||
| pH of the mobile phase | 1.95 | 100.36 | 0.61 | |
| 2.05 | 99.86 | 0.53 | ||
| Method II | Flow rate of the mobile phase | 0.90 mL·min−1 | 100.35 | 0.24 |
| 1.10 mL·min−1 | 99.83 | 0.17 | ||
| Column temperature | 20°C | 100.01 | 0.19 | |
| 30°C | 99.90 | 0.31 | ||
| Ethanol content of the mobile phase | 33% | 99.99 | 0.82 | |
| 37% | 100.43 | 0.57 | ||
| pH of the mobile phase | 1.95 | 100.29 | 0.63 | |
| 2.05 | 99.71 | 0.55 |
3.8 Application of chromatographic methods to APX quantification in pharmaceutical formulations and comparison of results
Six tablets (Eliquis), each containing 5 mg of APX, were analyzed by chromatographic methods. The results obtained by both chromatographic methods and the mean, SD, and RSD values calculated over six replications are given in Table 7. A comparison of the results obtained with both chromatographic methods in terms of mean was made using Student’s (t) test, and comparisons in terms of SDs were made using Fisher’s (F) test. When the results in the table were examined, it is clear that there was no significant difference between the two chromatographic methods in terms of accuracy and precision. The t and F values calculated for six trials were lower than the values reported in the relevant tables.
Statistical evaluation of analysis results of APX tablets (Eliquis, 5 mg)
| Sample | Method I | Method II | ||
|---|---|---|---|---|
| (mg·tablet−1) | (%) | (mg·tablet−1) | (%) | |
| 1 | 4.962 | 99.14 | 5.043 | 100.83 |
| 2 | 4.937 | 98.64 | 4.989 | 99.75 |
| 3 | 5.027 | 100.44 | 5.022 | 100.41 |
| 4 | 5.063 | 101.16 | 4.996 | 99.89 |
| 5 | 4.989 | 99.68 | 5.001 | 99.99 |
| 6 | 5.053 | 100.96 | 4.957 | 99.11 |
| Average | 5.005 | 100.00 | 5.001 | 100.00 |
| SD | 0.05 | 1.01 | 0.03 | 0.59 |
| RSD (%) | 1.01 | 1.01 | 0.59 | 0.59 |
| t value/t table | 0.1383/2.5706 | |||
| F value/F table | 2.9891/5.0503 | |||
3.9 Solution stability
There was no stability-related problem observed during the course of the analysis under different conditions. The test and working standard solutions showed good stability at the laboratory temperature for 48 h, 4°C (refrigerator temperature) for 10 days. The stability of the analytical solution was expressed as average percent recoveries, which were found to be in the range of 99.66–99.95%. The stability results in this study were found to be within the acceptable limits (±2%), which suggests that the standard solution can be evaluated under a normal laboratory environment without any significant loss. The solution stability results are depicted in Table 8.
Solution stability data at different storage conditions (n = 3)
| Analyte | Storage conditions | Average recovery (%) |
|---|---|---|
| APX | Normal laboratory temperature (25°C) for 12 h | 99.66 |
| Normal laboratory temperature (25°C) for 24 h | 99.72 | |
| Normal laboratory temperature (25°C) for 36 h | 99.74 | |
| Normal laboratory temperature (25°C) for 48 h | 99.83 | |
| Refrigerator temperature (4°C) for 10 days | 99.95 |
3.10 Greenness assessment of the chromatographic methods
The greenness evaluation pictograms of the chromatographic methods are presented in Figure 5. The score of the chromatographic Method I (using acetonitrile in the mobile phase) is 0.67, while the score of the chromatographic Method II (using ethanol in the mobile phase) is 0.74. In the AGREE pictogram of the chromatographic Method I, the scores corresponding to GAC principles 1, 8, and 11 are quite low, while the performance for principles 2, 4, 6, and 9 is excellent (Figure 5a). In the AGREE pictogram of the chromatographic Method II, the scores for GAC principles 1 and 8 are quite low, while the performance for principles 2, 4, 6, 9, and 11 is excellent (Figure 5c). It can be inferred that both chromatographic methods are green but the second chromatographic method (using ethanol in the mobile phase) is greener than the other method.
(a) AGREE pictogram of the chromatographic Method I. (b) Corresponding color scale for reference. (c) AGREE pictogram of the chromatographic Method II. 1 – direct analytical techniques should be used to avoid sample preparation. 2 – a minimum sample size and a minimum number of samples should be used. 3 – in situ measurements should be performed. 4 – analytical processes and procedures to be integrated to save energy and reduce the use of reagents. 5 – automated and miniaturized methods should be selected. 6 – derivatization should be avoided. 7 – high-volume waste generation should be avoided and waste should be managed appropriately. 8 – multi-analyte or multi-parameter methods should be preferred over methods using one analyte at a time. 9 – energy use should be minimized. 10 – reagents from renewable resources should be preferred. 11 – toxic reagents should be eliminated or replaced. 12 – operator safety should be improved.
4 Discussion
There is no reported green HPLC method for APX quantification in pharmaceutical products in the literature review. Only one green HPTLC method has been reported for this [34]. In this study, an HPLC method has been developed for the quantification of APX in pharmaceutical products using an environmentally and operator-friendly mobile phase, meeting all the needs of the validation process, without compromising the quality of chromatographic performance.
Since the greenness of analytical methods is evaluated from the sample preparation stage to the detection stage, we avoided the use of toxic chemicals during the sample preparation stage of the chromatographic method we developed. The use of ethanol both in the sample preparation phase and in the mobile phase has brought an alternative perspective to environmentally friendly analysis. It was compared with the analytical methods reported for the determination of the amount of APX in pharmaceutical products and the results are presented in Table 9. In all of the reported methods, acetonitrile was used as an organic modifier both in the sample preparation and in the mobile phase of HPLC analysis. We used ethanol in both stages.
Comparison of proposed and reported methods
| Reference | Mobile phase | Flow rate (mL·min−1) | Column | Retention time (min) | Range (μg·mL−1) | Run time (min) | Detection | LOD/LOQ | Precision (RSD %) |
|---|---|---|---|---|---|---|---|---|---|
| [27] | Water/acetonitrile (40:60) | 0.7 | Cosmosil C18 (250 mm × 4.6 mm; 5 µm) | 3.02 | 5–30 | 7 | 279 | 0.09/0.29 | 0.400 |
| [28] | 0.01 M sodium acetate (pH 4.5)/ACN (55:45) | 1.0 | INERTSIL ODS-2 C18 (250 mm × 4.6 mm × 5 µm) | 5.20 | 1–3 | 15 | 280 | 0.30/0.60 | <2.000 |
| [29] | Ammonium formate buffer/acetonitrile (65:35) | 1.1 | Zorbax C18 (150 mm × 4.6 mm, 5 μm) | 4.00 | 50–300 | 7 | 280 | N.D. | 0.470 |
| [30] | Phosphate buffer/acetonitrile | 1.3 | Ascentis C18 (100 mm × 4.6 mm, 2.7 µm) | 6.10 | 20–90 | 40 | 225 | 0.289/0.875 | 0.069 |
| [31] | 0.03 M ammonium acetate/acetonitrile (90:10 v/v) | 1.0 | Zorbax RX18, (250 mm × 4.6 mm, 5 μm) | 13.1 | 0.100–1.95 | 40 | 280 | 0.032/0.102 | 1.890 |
| [32] | Mobile phase | 1.0 | Ascentis Express C18 (4.6 mm × 100 mm, 2.7 µm) | 8.08 | 0.014–0.038% | 40 | 225 | 0.025/0.080 | 2.18 |
| A: 0.02 M ammonium dihydrogen orthophosphate buffer | |||||||||
| B: Acetonitrile | |||||||||
| [33] | The mobile phase A: formic acid solution B: Acetonitrile. Gradient conditions | 1.0 | LiChroCART Purospher Star C18; (55 mm × 4 mm, 3 μm) | 4.50 | 0.017–5.28 | 6.00 | 278 | 0.006/0.017 | 2.77 |
| Proposed method | Trifluoroacetic acid solution (0.1%)/ethanol (65:35, v/v) | 1.0 | Agilent C18 (250 mm × 4.6 mm, 5 μm) | 4.51 | 5–30 | 10 | 276 | 0.50/1.60 | 0.100 |
Since some of the reported HPLC methods [30,31,32] determine impurities and decomposition products, the analysis times are quite long, such as 40 min. This will cause the formation of an excessive amount of waste. Some of the reported HPLC methods [30,31,32] have very low sensitivity, and the % RSD values are above 2. However, the reported analytical methods are not green compared to the proposed method. To evaluate the green character of the developed methods and to compare them, a green assessment tool was applied. It was observed that the developed method is greener in terms of solvent and reagent hazards.
5 Conclusions
Developing environmentally friendly methods to prevent environmental pollution, reducing energy consumption, and waste management have become even more critical for the future of humanity. With this in mind, a green HPLC method was developed for the determination of the amount of APX in pharmaceutical products, using an environmentally and operator-friendly mobile phase without compromising the chromatographic performance quality. The developed green HPLC method met all the requirements of the validation process according to ICH guidelines, and it was observed to be linear, accurate, precise, robust, and sensitive. Safe and economical organic solvents such as ethanol were used in both sample preparation and detection stages of the developed method. In addition, the greenness profile score of the developed method is higher than the published chromatographic methods. For this reason, it is proposed that the developed method can be considered an environmentally friendly and economical alternative to the methods currently used for the safety of analysts and the environment in the quantitative analysis of APX in pharmaceutical products.
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Funding information: The authors state that there was no funding involved.
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Author contributions: İbrahim Bulduk: conceptualization, methodology, data curation, formal analysis, investigation; validation; writing original draft (equal); Erten Akbel: investigation (equal); methodology (equal); data curation (equal); writing – review and editing (equal); software (equal); Süleyman Gökçe: formal analysis (equal); investigation (equal); validation (equal); investigation visualization (equal); software (equal); writing – original draft (equal).
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Conflict of interest: The authors state that there was no funding involved.
References
[1] Görög S. The changing face of pharmaceutical analysis. TrAC Trends Anal Chem. 2007;26:12–7. 10.1016/j.trac.2006.07.011.Search in Google Scholar
[2] Snyder LR, Kirkland JJ, Dolan JW. Introduction to modern liquid chromatography. Hoboken, NJ, USA: John Wiley & Sons; 2009.10.1002/9780470508183Search in Google Scholar
[3] Welch CJ, Wu N, Biba M, Hartman R, Brkovic T, Gong X, et al. Greening analytical chromatography. TrAC Trends Anal Chem. 2010;29:667–80. 10.1016/j.trac.2010.03.008.Search in Google Scholar
[4] ICH Harmonised Guideline. Impurities: Guideline for residual solvents Q3C (R7). [Internet]. 2018. https://fdocuments.us/document/impurities-guideline-for-residual-solvents-residual-solvents-in-drug-substances.html?page=1.Search in Google Scholar
[5] Sheldon RA. Fundamentals of green chemistry: Efficiency in reaction design. Chem Soc Rev. 2012;41:1437–51. 10.1039/C1CS15219J.Search in Google Scholar
[6] Tobiszewski M, Mechlińska A, Namieśnik J. Green analytical chemistry—theory and practice. Chem Soc Rev. 2010;39(8):2869–78.10.1039/b926439fSearch in Google Scholar PubMed
[7] Tobiszewski M, Marć M, Gałuszka A, Namieśnik J. Green chemistry metrics with special reference to green analytical chemistry. Molecules. 2015;20:10928–46. 10.3390/molecules200610928.Search in Google Scholar PubMed PubMed Central
[8] Gałuszka A, Migaszewski ZM, Konieczka P, Namieśnik J. Analytical eco-scale for assessing the greenness of analytical procedures. TrAC Trends Anal Chem. 2012;37:61–72. 10.1016/j.trac.2012.03.013.Search in Google Scholar
[9] Keith LH, Gron LU, Young JL. Green analytical methodologies. Chem Rev. 2007;107:2695–708. 10.1021/cr068359e.Search in Google Scholar PubMed
[10] Tobiszewski M. Metrics for green analytical chemistry. Anal Methods. 2016;8:2993–9. 10.1039/C6AY00478D.Search in Google Scholar
[11] Capello C, Fischer U, Hungerbühler UK. What is a green solvent? A comprehensive framework for the environmental assessment of solvents. Green Chem. 2007;9:927–34. 10.1039/B617536H.Search in Google Scholar
[12] Plotka J, Tobiszewski M, Sulej AM, Kupska M, Górecki T, Namieśnik J. Green chromatography. J Chromatogr A. 2013;1307:1–20. 10.1016/j.chroma.2013.07.099.Search in Google Scholar PubMed
[13] Miyabe K, Takeuchi S, Tezuka Y. Adsorption characteristics in reversed-phase liquid chromatography using ethanol/water mixed solvent. Adsorption. 1999;5:15–24. 10.1023/A:1026477619947.Search in Google Scholar
[14] Ribeiro RL, Bottoli CB, Collins KE, Collins CH. Reevaluation of ethanol as organic modifier for use in HPLS-RP mobile phases. J Braz Chem Soc. 2004;15:300–6. 10.1590/S0103-50532004000200022.Search in Google Scholar
[15] Shen Y, Chen B, van Beek TA. Alternative solvents can make preparative liquid chromatography greener. Green Chem. 2015;17:4073–81. 10.1039/C5GC00887E.Search in Google Scholar
[16] Mohamed HM. Green, environment-friendly, analytical tools give insights in pharmaceuticals and cosmetics analysis. TrAC Trends Anal Chem. 2015;66:176–92. 10.1016/j.trac.2014.11.010.Search in Google Scholar
[17] Shaaban H, Górecki T. Current trends in green liquid chromatography for the analysis of pharmaceutically active compounds in the environmental water compartments. Talanta. 2015;132:739–52. 10.1016/j.talanta.2014.09.050.Search in Google Scholar PubMed
[18] Luettgen JM RM, Knabb RM, He K, Pinto DJP, Rendina AR. Apixaban inhibition of factor Xa: microscopic rate constants and inhibition mechanism in purified protein systems and in human plasma. J Enzyme Inhib Med Chem. 2011;26(4):514–26.10.3109/14756366.2010.535793Search in Google Scholar PubMed
[19] Jiang X, Crain EJ, Luettgen JM, Schumacher WA, Wong PC. Apixaban, an oral direct factor Xa inhibitor, inhibits human clot-bound factor Xa activity in vitro. Thromb Haemost. 2009;101(4):780–2. 10.1160/TH08-07-0486.Search in Google Scholar
[20] Eliquis (2.5 mg film-coated tablets) Prescribing Information. Princeton, NJ and New York, NY: Bristol-Myers Squibb and Pfizer [Internet]. 2022, https://labeling.pfizer.com/ShowLabeling.aspx?id=12118.Search in Google Scholar
[21] Tantawy MA, El-Ragehy NA, Hassan NY, Abdelkawy M. Stability-indicating spectrophotometric methods for determination of the anticoagulant drug APX in the presence of its hydrolytic degradation product. Spectrochim Acta A. 2016;159:13–20. 10.1016/j.saa.2016.01.029.Search in Google Scholar PubMed
[22] Mahendra B, Sundari KH, Vimalakkannan T. Method developed for the determination of APX by using UV spectrophotometric. Int J Res Pharm Chem Anal. 2019;1:83–7. 10.33974/ijrpca.v1i3.115.Search in Google Scholar
[23] Radhika AG, Singh A, Sowmya A, Akiful HM, Bakshi V, Boggula N. Comparative studies of APX in bulk and its formulations by Uv-Spectroscopy (Zero Derivatives and Area Under Curve). Int J Pharm Biol Sci. 2018;8:1002–8.Search in Google Scholar
[24] Anusha K, Sowjanya G, Ganapaty S. Development and validation of UV spectrophotometric methods for apixaban in tablets. Eur J Biomed Pharm Sci. 2018;5:929–33.Search in Google Scholar
[25] El-Bagary RI, Elkady EF, Farid NA, Youssef NF. Validated spectrofluorimetric methods for the determination of APX and tirofiban hydrochloride in pharmaceutical formulations. Spectrochim Acta Part A. 2017;174:326–30. 10.1016/j.saa.2016.11.048.Search in Google Scholar PubMed
[26] Rizk M, Sultan MA, Taha EA, Attiab AK, Abdallah YM. Sensitive validated voltammetric determination of APX using a multi-walled carbon nanotube-modified carbon paste electrode: Application to a drug product and biological sample. Anal Methods. 2017;9:2523–34. 10.1039/C7AY00244K.Search in Google Scholar
[27] Gholap SV, Mankar SD, Dighe SB. Analytical method development and validation of Apixaban by RP-HPLC method. Int J Anal Exp Modal Anal. 2020;6(12):1817–44.Search in Google Scholar
[28] Chitale AS, Hamrapurkar P. Development and validation of assay method for estimation of Apixaban in bulk drugs and its marketed formulation. Int J Adv Res Ideas Innov Technol. 2018;4(6):367–70.Search in Google Scholar
[29] Rajput RS, Lariya N. A stability indicating method development and validation of apixaban in pharmaceutical dosage form by using RP-HPLC and in-vitro evaluation of apixaban suspension delivery through enteral feeding tubes. J Med Pharm Allied Sci. 2022;11(1):4358–63. 10.22270/jmpas.V11I1.2195.Search in Google Scholar
[30] Al-Ani I, Hamad M, Al-Shdefat R, Mansoor K, Glogor F, Dayyish WA. Development and validation of stability indicating RP-HPLC method of apixaban in commercial dosage forms. Int J Pharm Sci Res. 2019;12(1):241–51. 10.13040/IJPSR.0975-8232.Search in Google Scholar
[31] Subramanian VB, Katari NK, Dongala T, Jonnalagadda SB. Stability indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms robustness study by quality by design approach. J Biomed Chromatogr. 2019;34(1):1–22. 10.1002/bmc.4719.Search in Google Scholar PubMed
[32] Landge SB, Jadhav SA, Dahale SB, Solanki PV, Bembalkar SR, Mathad VT. Development and validation of stability indicating RP-HPLC method on the core-shell column for determination of degradation and process related impurities of apixaban – an anticoagulant drug; American. J Anal Chem. 2015;6(6):1–11. 10.4236/ajac.2015.66052.Search in Google Scholar
[33] Gouveia F, Bicker J, Santos J, Rocha M, Alves G, Falcão A, et al. Development, validation and application of a new HPLC-DAD method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma. J Pharm Biomed Anal. 2020;181:113109. 10.1016/j.jpba.2020.113109.Search in Google Scholar PubMed
[34] Prajapati P, Rajpurohit P, Pulusu VS, Shah S. Green and sustainable analytical chemistry-driven chromatographic method development for stability study of apixaban using box–behnken design and principal component analysis. J Chromatogr Sci. 2023;6:1–11. 10.1093/chromsci/bmad033.Search in Google Scholar PubMed
[35] Delavenne X, Mismetti P, Basset TJ. Rapid determination of apixaban concentration in human plasma by liquid chromatography/tandem mass spectrometry: Application to pharmacokinetic study. Pharm Biomed. 2013;78–79:150–3. 10.1016/j.jpba.2013.02.007.Search in Google Scholar PubMed
[36] Lindahl S, Dyrkorn R, Spigset O, Hegstad S. Quantification of apixaban, dabigatran, edoxaban, and rivaroxaban in human serum by UHPLC-MS/MS—method development, validation, and application. Ther Drug Monit. 2018;40:369–76. 10.1097/FTD.0509.Search in Google Scholar
[37] Țilea I, Popa DS, Xantus TS, Primejdie D, Grigorescu B, Tileka B, et al. Determination of apixaban levels in human plasma by a high-throughput liquid chromatographic tandem mass spectrometry assay. Rev Romana de Med de Lab. 2015;23:115–25. 10.1515/rrlm-2015-0006.Search in Google Scholar
[38] Jeong HC, Kim TE, Shin KH. Development and validation of LC-MS/MS method for determination of plasma apixaban. Trans Clin Pharmacol. 2019;27:33–41. 10.1556/1326.2021.00948.Search in Google Scholar
[39] Derogis PBM, Sanches LR, de Aranda VF, Colombini MP, Mangueira CLP, Katz M, et al. Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS). PLoS ONE. 2017;12(2):e0171272. 10.1371/journal.pone.0171272.Search in Google Scholar PubMed PubMed Central
[40] Validation of analytical procedures: text and methodology Q2(R1)-ICH harmonized tripartite guideline. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2005.Search in Google Scholar
[41] CDER. Center for drug evaluation and research. Reviewer guidance: Validation of chromatographic methods. [Internet]. 1994 https://www.gmp-compliance.org/files/guidemgr/1-6-13.pdf.Search in Google Scholar
[42] Płotka-Wasylka J. A new tool for the evaluation of the analytical procedure: Green analytical procedure index. Talanta. 2018;181:204–9.10.1016/j.talanta.2018.01.013Search in Google Scholar PubMed
[43] Pena-Pereira F, Wojnowski W, Tobiszewski M. AGREE—Analytical greenness metric approach and software. Anal Chem. 2020;92:10076–82.10.1021/acs.analchem.0c01887Search in Google Scholar PubMed PubMed Central
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