Abstract
A straightforward, dependable, and quick RP-LC method for the analysis of abiraterone acetate in its dose form and human urine has been devised. With DAD detection, sensitivity was reported to be high. The LOD and LOQ of the procedure were deemed adequate. The suggested approach was exhaustively validated in accordance with ICH requirements, and the findings demonstrated that it was exact, accurate, selective, and sensitive for the analysis of this pharmaceutical. The chromatographic separation was realized using a X-Terra RP-18 (150 × 4.60 mm i.d. × 5 μm) column and a UV detector set at 255 and 267 nm. In addition, pKa values were calculated based on the relationship between the retention factor and the pH of the mobile phase. The influence of the composition of the mobile phase on the ionization constant was investigated by measuring the pKa at various acetonitrile–water combinations ranging from 50 to 70% (v/v).
1 Introduction
Prostate cancer is the second leading cause of cancer-related mortality among men, after lung cancer [1]. Cancer of the prostate is primarily a hormone-dependent tumor. The production of testosterone, which promotes tumor growth, is inhibited by ADT. After chemotherapy, the conventional use of abiraterone acetate for prostate cancer is to treat castration-resistant prostate cancer [2].
Abiraterone acetate is a derivative of steroidal progesterone (Fig. 1). A selective inhibitor of the testosterone-producing enzymes cytochrome P450 17x-hydrolyase/17 and CYP17, CYP complex [3–5]. Abiraterone acetate is classified as an adrenal inhibitor and is used to treat advanced prostate and breast cancer with hormone therapy [6]. Male hormones (testosterones/androgens) promote the development of prostate cancer. Zytiga, a tablet formulation containing Abiraterone acetate and prednisone, has been granted approval by both the US-FDA and EMA for the treatment of metastatic castration-resistant prostate cancer (mCRPC) in males who have previously undergone initial chemotherapy with docetaxel or ketakonazole [7, 8].
Chemical structure of abiraterone acetate
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01173
Since it aids in the explanation of chemical processes like chemical absorption, distribution, and elimination, the acid-base dissociation constant, or pKa, has long been recognized as an important metric in research on ADME and toxicity. This is because it can explain chemical phenomena such as how chemicals are absorbed, distributed, and eliminated. This notable variable possesses numerous applications within research domains, including but not limited to the advancement of medicinal compounds, solvent separation, acid-base volumetric analysis, and ion conveyance. The toxicity, chromatographic retention behavior, and medicinal qualities of organic acids and bases are influenced by their acid-base characteristics. Pharmaceutical compounds are commonly classified as either weak acids or weak bases, and their absorption primarily occurs through passive diffusion of the non-ionized component. Hence, it holds significant importance to ascertain the ionization state of a drug at a specific pH. The pharmacokinetic behavior of a drug compound within an organism is contingent upon the dissociation constant of said compound. Therefore, accurate determination of the drug dissociation constant of a pharmaceutical compound is crucial. Furthermore, while RP-LC separation methods have employed mobile phases consisting of acetonitrile (ACN) and water, the pKa of abiraterone in binary mixtures of ACN and water remains undetermined. Only two prediction programs values could be found for abiraterone [9, 10].
There have been numerous publications on the determination of this drug, including the use of HPLC [11–16], liquid chromatography–tandem mass spectrometric technique (LC–MS/MS) [17–20], and ultra-performance liquid chromatography (UPLC) [21].
The utilization of RP-LC has been established as a potent method for the determination of dissociation constants. This is attributed to its ability to operate with minimal quantities of compounds, its capacity to analyze impure samples, and its ability to overcome the limitations of poor water solubility, as reported in references [22–24]. This methodology necessitates solely the retention times, without the need for measuring solute or titrant concentrations. Furthermore, the computation process is uncomplicated and unaffected by the level of impurities in the solute.
The present study is centered on the assessment of pKa values of abirateron in various ACN-water mixtures, namely 50, 60, and 70% v/v. The objective of this research is to address the dearth of knowledge pertaining to the acid-base equilibria of such compounds through chromatographic measurements.
Despite the widespread use of abiraterone acetate in the treatment of prostate cancer, there is currently no published methodology for the determination of abiraterone acetate in human urine samples using RP-LC techniques with UV detection, as far as our knowledge extends. Abiraterone was successfully determined in both dosage form and human urine sample using the optimal separation conditions obtained from pKa studies.
2 Experimental section
2.1 Chemicals and reagents
The chemicals were utilized without undergoing additional purification procedures. Abiraterone acetate and everolimus (used as an internal standard) were procured from Sigma-Aldrich (St. Louis MO, USA).
The organic modifier utilized in this study was HPLC grade acetonitrile, which was sourced from Merck in Darmstadt, Germany. The ortho-phosphoric acid utilized in this study, with a minimum purity of 85%, was procured from Riedel (Riedel-de Haen, Seelze, Germany). The chemicals utilized in this study were sodium hydroxide (NaOH), hydrochloric acid (HCl), uracil, and methanol, which were procured from Sigma-Aldrich (St. Louis, MO, USA).
2.2 Apparatus
An Agilent 1260 series HPLC system equipped with a ternary solvent pump, online degasser, automatic injection system, column heater, and multi-wavelength detector was utilized to perform the LC analysis. Abiraterone acetate and everolimus were subjected to UV detection at wavelengths of 255 and 267 nm, respectively. The experiment was conducted with a flow rate of 1 mL min−1, utilizing an X-Terra RP-18 (150 × 4.60 mm i.d. × 5 μm) column as the stationary phase at a temperature of 25 °C. The pH measurements were conducted using a Mettler Toledo MA 235 pH/ion analyzer in conjunction with a Hanna HI 1332 Ag/AgCl combined glass electrode. The selection of Everolimus as the internal standard was made.
2.3 Chromatographic procedure
In the course of this investigation, the mobile phases that were analyzed consisted of ACN-water blends with volumetric ratios of 50%, 60%, and 70%, respectively, and were supplemented with 10 mM o-phosphoric acid. The pH range of the mobile phase was modulated from 2.5 to 7.0 through the incorporation of sodium hydroxide. The injection volume of 20 μL was maintained at a flow rate of 1.0 mL min−1.
Prior to the initial injection, the column underwent a pre-conditioning process for a minimum of one hour at a low flow rate of 0.5 mL min−1, utilizing the mobile phase at the appropriate pH. Retention time values (tR) for each compound were determined through three distinct injections for every mobile phase composition and pH that was taken into account.
The pH of the mobile phase exerts a significant influence on the chromatographic retention of ionizable compounds. Efficient separations of ionizable compounds by RP-LC often necessitate precise measurement and regulation of the mobile phase pH [25].
Various techniques can be employed for the determination of the pH of the mobile phase [26]. Prior to combining the aqueous buffer with the organic modifier, the prevalent approach involves determining the buffer's pH. A more stringent methodology, as advised by the IUPAC, was employed, wherein the pH of the mobile phase was determined subsequent to the combining of the aqueous buffer and organic modifier. The electrode system utilized for pH measurement in this scenario can be calibrated through the utilization of either aqueous buffers or buffers that have been prepared in the same solvent composition employed as the mobile phase. Acquiring knowledge pertaining to the pH value of reference buffers that are formulated in diverse aqueous-organic solvent mixtures is necessary [27].
Equation (1) is utilized by NLREG to iteratively refine the parameters, resulting in a minimized sum of square residuals (Um) for the purpose of determining the dissociation constants of the compounds under investigation. The equation for ns involves the variables ki,exp and ki,calc, which respectively represent the experimental and calculated values of the retention factor for a given solution i.
2.4 The process of creating standard solutions and developing a calibration plot
A standard stock solution of the drug was prepared through dissolution in MeOH at a concentration of 100 μg mL−1. The preparation of standard solutions for the analysis of pharmaceutical dosage forms involved the use of abiraterone acetate and everolimus, which were dissolved in the mobile phase. The concentrations of these solutions were adjusted within the range of 1.0–20 μg mL−1. The concentration of drugs in urine was subjected to analysis within the range of 1.0–40 μg mL−1. The construction of calibration curves for RP-LC analysis involved plotting the drug-to-IS peak area ratio against the drug concentration. A stable concentration of IS was sustained at 20.0 μg mL−1. Each solution underwent five injections of 20 µL, and the resulting peak area ratio of each drug with respect to the internal standard was graphed against the corresponding concentration to generate a calibration graph. All solutions were shielded from light and utilized within a 24 h period. The measurement of dead time was conducted through the injection of a uracil solution.
The study assessed the ruggedness and precision of the measurements on separate occasions, distinguishing between within-day (n = 5) and between-day (n = 5) variations. The robustness and precision of the method were evaluated by calculating the relative standard deviations [29–31]. The quantitative depiction of the precision and accuracy of the developed techniques is achieved through the utilization of Bias% (relative error).
2.5 The evaluation of pharmaceutical dosage forms
The pharmaceutical formulation of abiraterone acetate, specifically Zytiga 250 mg, was procured from a nearby pharmacy. A total of 20 tablets were subjected to individual weighing, and subsequently, their average weight was determined and pulverized. A quantity of tablet powder, corresponding to 100 mg of abiraterone acetate, was introduced into a 100 mL volumetric flask. Subsequently, 50 mL of mobile phase was added to the flask, and the mixture was subjected to sonication for a duration of 30 min at a controlled temperature to facilitate the dissolution of the powder. The resulting solution was then brought to volume with the same solvent. Subsequent to the aforementioned procedure, extract precisely 1.0 mL of the specimen solution and transfer it into a volumetric flask with a capacity of 10 mL. Proceed to dilute the solution with the mobile phase until it reaches the mark on the flask, ensuring that it is thoroughly mixed. The clear filtrate was divided into suitable aliquots, and the appropriate internal standard (everolimus) solution was added to generate appropriate solutions. The quantity of the substance was determined using the respective regression formula.
2.6 Analysis of spiked urine samples
The urine specimens were collected from asymptomatic participants in a timely manner prior to the commencement of the experimental procedures. The urine sample was divided into equal volumes and transferred into a flask that had been calibrated for accuracy. A specific volume of urine sample was supplemented with a drug that was dissolved in ultrapure water, resulting in a final concentration of 100 μg mL−1. The quantity of internal standard incorporated into the specimens. The drug concentration was altered within the range of 1–40 μg mL−1 in urine samples obtained from human subjects. The solutions underwent filtration and analysis through RP-LC methodology.
3 Results and discussions
3.1 Determination of dissociation constants (pKa)
Determining the dissociation constants (pKa) of drugs is a fundamental step in pharmaceutical research. Accurate knowledge of pKa values aids in drug design, formulation development, understanding pharmacokinetics, optimizing drug-target interactions, and ensuring therapeutic efficacy and safety. By comprehensively characterizing the ionization behavior of drugs, researchers can make informed decisions that contribute to the successful development of effective and safe medications.
It is customary in contemporary drug development to produce compounds that exhibit high lipophilicity and moderate aqueous solubility. The insolubility of pharmaceuticals in water is frequently considered a disadvantage when evaluating their physicochemical characteristics. The solubility of the drugs under investigation can be enhanced by the addition of an organic co-solvent to the aqueous mobile phase, as reported in previous studies [32, 33]. The present investigation employed LC technology to examine the correlation between the retention of abiraterone and the pH of the mobile phase. Empirical data were employed to determine the pKa values and intrinsic capacity factors. The pKa values were determined by analyzing k/pH data pairs with the aid of the NLREG software [28]. The pKa value of abiraterone is summarized in Table 1.
The pKa values of abiraterone obtained by chromatographic method in ACN–water media at 25 °C
| Prediction programs | Yasuda–Shedlovsky | XACN | NLREG | |||
| 50% (v/v) ACN | 60% (v/v) ACN | 70% (v/v) ACN | ||||
| Abiraterone | 4.81* 5.50** | 4.35 | 4.78 | 4.13 ± 0.09 | 3.90 ± 0.08 | 3.72 ± 0.09 |
* http://www.chemicalize.org. (Accessed 26 April 2023).
** Pan X, Wang H, Li C, et al. MolGpka: A Web Server for Small Molecule pK(a) Prediction Using a Graph-Convolutional Neural Network. J Chem Inf Model 2021; 61(7): 3159–3165. doi:10.1021/acs.jcim.1c00075.
The obtained curves exhibited typical sigmoidal patterns, indicating the correlation between the pH of the mobile phase and the capacity factors of the analyte. Figure 2 illustrates the instances of abiraterone acetate present in the mobile phase, which comprises 70% ACN-water (v/v).
Capacity factors of the abiraterone are plotted as a function of the mobile phase pH using NLREG program
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01173
The logarithm of the water molar concentration in a solvent combination is denoted by log[H2O], while the dielectric constant of the mixture is denoted by ε. The values of ε for various ACN-water mixtures were calculated by Barbosa et al. [36]. The values of the substance under study were calculated using linear equations created by plotting pKa + log[H2O] data against 1/ε values. If you plot pKa versus 1/ε, you should be able to extrapolate to pure water to get the aqueous pKa of the compounds [33]. The second method is based on the linear connections between the pKa values of the chemicals examined and the mole fraction of ACN (XACN) at 25 °C. The dissociation process is regulated not just by electrostatic interactions but also by particular solute-solvent interactions, which may account for variations in pKa values. Electrostatic interactions are more powerful than solute-solvent interactions, which explains why this is the case [37]. The intercept of linear equations gives the values of the thermodynamic pKa of the substances in question when dissolved in water. Table 1 displays the pKa values calculated using the two different approaches.
As can be seen in Fig. 3, the plots follow a linear pattern with a range of slopes. These straight lines may be clarified and made clearer by factoring in the structural properties of the ACN + water mixtures. Figure 3 displays Y–S graphs for researched chemotherapeutic drugs that comprise basic functional groupings. Both figures show that the slopes of the main functional categories are all negative.
Yasuda–Shedlovsky and mole fraction plots for the abiraterone
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01173
3.2 Quantitative determination
The present study aimed to determine the optimal chromatographic conditions for RP-LC analysis of drugs. To achieve this, the impact of pH variations in the mobile phase and column temperature was investigated. Multiple pH values ranging from 2.5 to 7.0 were evaluated, and after careful analysis, a pH of 2.5 was determined to be the optimal value based on its superior peak asymmetry and retention values. The pH of the mobile phase has consistently been modified by utilizing a concentration of 10 mM orthophosphoric acid. The temperature of the column was maintained within the range of 25–30 °C. Research has demonstrated that elevating the temperature can mitigate the phenomenon of drug tailing. The selection of 25 °C was based on the advantages of reduced analysis time and enhanced peak morphology. Various mobile phase compositions ranging from 50 to 70% (v/v) were evaluated. The optimal carrier for RP-LC analysis was determined to be a mobile phase consisting of ACN and water in a 70:30 (v/v) ratio, with the addition of 10 mM H3PO4 at pH 2.5 and 4 for pharmaceutical dosage forms and urine, respectively. The flow rate was set at 1.0 mL per minute.
Using the conditions described above, a satisfactory chromatographic peak resolution was obtained in a short analysis time (4 min).
3.3 Analysis of a spiked urine sample
The method was verified in accordance with the ICH recommendations for the validation of analytical methods [30, 31, 38]. Table 2 presents the validation data pertaining to the linearity range, regression equations, correlation coefficient, LOD, and LOQ values for pharmaceutical dosage forms and urine analysis. The quantitation of the studied compound was performed by utilizing the ratio of the peak area of the compound to the internal standard (IS) against its concentration.
Statistical evaluation of the calibration data of abiraterone acetate for urine and pharmaceutical analysis
| Urine | Pharmaceutical dosage form | |
| Linearity range (µg mL−1) | 1.0–40 (n = 5) | 1–20 (n = 5) |
| Slope | 0.0397 | 0.0392 |
| Intercept | 0.0211 | 0.0363 |
| Correlation coefficient (r) | 0.999 | 0.999 |
| Detection limit (LOD) (µg mL−1) | 0.099 | 0.074 |
| Quantitation limit (LOQ) (µg mL−1) | 0.300 | 0.223 |
The precision and reproducibility of the method were assessed through the analysis of standard solutions in the mobile phase, with replicates being performed. Table 3 presents a summary of the mean recovery and RSD, which were utilized to characterize the repeatability and reproducibility. Table 3 indicates that there was no statistically significant difference observed for the assay, as determined through within-day and between-day testing. The RSD percentage values ranged from 0.111 to 0.486 for the concentrations under investigation. The findings of this study indicate that the method exhibits a high degree of precision.
Summary of repeatability (intra-day) and reproducibility (inter-day) precision data for abiraterone acetate
| Concentration | Intra-day | Inter-day | |
| Mean Recovery* % ± RSD % | Mean Recovery* % ± RSD % | ||
| Dosage Form | 1 | 100.410 ± 0.351 | 100.773 ± 0.486 |
| 10 | 100.094 ± 0.111 | 100.120 ± 0.312 | |
| Urine | 5 | 100.621 ± 0.337 | 100.643 ± 0.422 |
| 20 | 99.989 ± 0.141 | 100.144 ± 0.171 | |
*Each value is obtained from five experiments (n = 5).
The method proposed here was employed for the analysis of drugs present in pharmaceutical dosage forms as well as in human urine specimens. The findings derived from the drug analysis demonstrate that the suggested approach is suitable for the quantification and regular quality assessment of drugs in commercial specimens. The observed disparities between the claimed quantities and the measured quantities were minimal, and the relative standard deviation (RSD%) values were deemed satisfactory. The label of abiraterone acetate indicated a quantity of 250.000 mg, whereas the measured amount was 249.873 mg, accompanied by a relative standard deviation of 0.095 based on a sample size of 5. Following five iterations of experimentation, the average percentage recoveries were determined to be 100.075, exhibiting RSD% values of 0.0595. To assess the suitability of the approach for biological specimens, recovery experiments were conducted for the quantification of drugs in human urine using the calibration curve technique, resulting in the attainment of the designated percentage recoveries. The investigated drug was added to urine samples that had no detectable levels of the substance. Following five iterations of experimentation, the average percentage recoveries for urine samples were determined to be 100.022, with relative standard deviation (RSD%) values of 0.035. Figure 4a and b indicate the customary chromatograms of a pharmaceutical dosage form and a urine sample spiked with the substance, respectively.
Typical chromatograms Pharmaceutical dosage form a) and for human urine b) spiked with abiraterone acetate (1), everolimus (2) (IS). (Chromatographic condition as: ACN and water in a 70:30 (v/v) ratio, with the addition of 10 mM H3PO4 at pH 2.5 and 4.0 for pharmaceutical dosage forms and urine)
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01173
The analysis of pharmacological dosage forms was conducted at a pH of 2.5, while urine analysis was carried out at a pH of 4.0 due to the presence of an unidentified peak occurring at around 1.5 min. Due to this factor, the retention times exhibit variation. The chromatogram acquired from the urine sample does not exhibit any insignificant peaks. The accuracy and precision of the pharmaceutical formulation and urine were determined through recovery studies utilizing the standard addition method subsequent to their analysis. The results indicate that the proposed method for determining abiraterone acetate in human urine is both reproducible and sensitive. This is evidenced by the high percentage of recovery data and low RSD% values, which suggest that the method is free from interference and easy to perform.
In the literature, there is no method for the determination of abiraterone acetate in human urine samples using RP-LC techniques with UV detection. Sankar et al. [16] determined abiraterone acetate in pharmaceutical dosage form and this method required 7.45 min for analysis time. For this method the limit of detection and the limit of quantitation was found to be 0.451 μg mL−1 and 1.369 μg mL−1 respectively. Reddy et al. [13] was developed RP-HPLC method for the determination assay of abiterone acetate in pharmaceutical with the analysis time of about 5 min. The limits of detection and quantification were 0.23 and 0.70 μg mL. The method described above provides a combination of faster analysis time and improved limits of detection.
4 Conclusions
The RP-LC method that has been put forth allows for the quantification of abiraterone acetate in both human urine samples and dosage forms. This method is characterized by its simplicity, speed, high sensitivity, accuracy, and precision. The X-Terra column facilitated an analysis time of less than 5 min for the compound under study. The report on validation affirms that the proposed technique is suitable as a standard method for detecting the drug in formulations, end products, or exposure investigations.
Furthermore, this manuscript introduces the first investigation pertaining to the determination of pKa values of abiraterone through the employment of RP-LC methodology in ACN-water binary mixtures. The determined pKa values are suitable for estimating the effect of eluent pH on retention.
Conflict of interest statement
The authors have declared that they have no conflicts of interest.
Acknowledgments
The authors are thankful for the financial aid received from the Scientific and Technological Research Council of Turkiye (TUBITAK). We gratefully acknowledge Dr. Jose Luis Beltran from the University of Barcelona for kindly providing the NLREG 4.0 program.
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