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Article

Analysis of Aspirin and Dipyridamole in a Modern Pharmaceutical Formulation-Drug Release Study and Permeability Assessment

by
Georgios Kamaris
1,†,
Nikoletta Pantoudi
1,†,
Konstantina Chachlioutaki
2 and
Catherine K. Markopoulou
1,*
1
Laboratory of Pharmaceutical Analysis, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Laboratory of Pharmaceutical Technology, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2025, 15(11), 6048; https://doi.org/10.3390/app15116048
Submission received: 29 March 2025 / Revised: 25 May 2025 / Accepted: 27 May 2025 / Published: 28 May 2025
(This article belongs to the Special Issue New Trends in the Development of Pharmaceutical Forms for Human and Veterinary Use)
Browse Figures
Versions Notes

Abstract

:
Oral administration of dipyridamole (DIP) with acetylsalicylic acid (ACA) is recommended in thromboembolic conditions or for the treatment of myocardial infarction and stroke. The present study presents an alternative dosage form of these two active ingredients, consisting of a honey core and a dark chocolate coating. The composition masks the bitter taste, is palatable and ensures compliance of a wide range of patients, mainly pediatric. For the simultaneous quantitative determination of the analytes, a Diode Array Detector/Fluorescence Detector (HPLC-DAD/FLD) method was used with a C18 column (250 mm × 4.6 mm, 5 μm) and an isocratic two-phase system (A: H2O 0.2% formic acid—B: Acetonitrile-H2O 90:10 v/v) 65:35 v/v. The method was validated according to ICH guidelines (r2 > 0.999, RSD < 2.3%, % Recovery > 95.4%), and a stability study of the two active ingredients as well as salicylic acid (SAL), which is a hydrolysis product of ACA, was followed. Finally, a digestion protocol (oral cavity–stomach–intestine) for edible materials was applied to determine the release rate of ACA, DIP and SAL in the gastrointestinal tract, while an in vitro permeability study (Papp) was subsequently performed in Franz cells. The results show satisfactory behavior of ACA and DIP and provide a trigger for further studies of the formulation.

1. Introduction

Cardiovascular diseases are among the leading causes of mortality worldwide [1]. The indicated pharmacological treatment for therapy and prevention of thromboembolic diseases includes the use of antiplatelet, anticoagulant, and thrombolytic (fibrinolytic) agents [2,3]. Aspirin (acetylsalicylic acid/ACA) is the most widely studied antiplatelet agent used (75–100 mg/day) in the acute phase and in the secondary prevention of ischemic stroke and cardiovascular disease, either alone or in combination therapy [4,5]. Similarly, dipyridamole (DIP) has been used as a vasodilatory, antianginal and antiplatelet agent, mainly for the secondary prevention of myocardial infarction and ischemic stroke. It is noteworthy that dipyridamole inhibits the transport of aspirin, thereby increasing its trapping in platelets, which explains their therapeutic synergy [6,7,8]. Thus, co-administration of 25–50 mg of aspirin (daily) and 200 mg (at least two times/day) of dipyridamole reduces the risk of stroke recurrence by 23%, compared to monotherapy [9,10]. Related pharmaceutical preparation is available on the market under the trade name Aggrenox®. Its dosage form is a hard gelatin capsule containing dipyridamole 200 mg in extended-release form and aspirin 25 mg in immediate-release form. It is indicated for patients who have suffered a transient ischemic attack or stroke due to thrombosis. This preparation is not currently approved by the FDA for administration to children under 11 years of age due to limited clinical studies. However, the use of a corresponding therapeutic regimen is usually recommended for preventive reasons, even in pediatric patients. According to the clinical practice guidelines of the American College of Chest Physicians, antiplatelet therapy with ACA (1 to 5 mg/kg) and DIP (2 to 5 mg/kg) is recommended for children with ventricular assist devices [11]. A similar treatment is also proposed for Kawasaki disease [12]. In fact, a study conducted at a children’s hospital in China shows that the combination of aspirin (3–5 mg/kg daily) and dipyridamole (25–50 mg three times per day), along with intravenous human immunoglobulin, significantly enhances the effectiveness of the treatment [13].
Furthermore, the lack of suitable formulations, especially for pediatric patients, is faced daily in hospitals around the world, leading to the conversion of existing commercial formulations (e.g., dipyridamole) into pediatric dosage forms [14]. As for aspirin, in addition to the conventional dosage forms, chewable gum (Aspergum®) is available in the United States, primarily intended for specific groups of patients with swallowing difficulties, such as children and the elderly [15]. These reasons have prompted the scientific community, in recent decades, to focus on the development of drug delivery systems, such as orodispersible tablets and films, chewable formulations and mini-tablets, suitable for the aforementioned patients [16]. A particularly easy-to-administer delivery system for active pharmaceutical ingredients (APIs) is honey. Honey not only has a sweet taste but also offers well-known beneficial effects and protective properties on various human systems, such as the cardiovascular, nervous, respiratory and gastrointestinal systems [17]. Remarkable are its antioxidant properties, due to the presence of flavonoids and phenolic compounds as well as its antimicrobial and preservative action [18]. The United States Food and Drug Administration (FDA) has approved several honey-based medicinal products, which mainly include gels, dressings, ointments, and pastes [17]. A similar innovative pharmaceutical form with honey and chocolate, which contains ibuprofen, is intended for pediatric patients, as the substance is a widely prescribed pediatric drug, but it has a bitter taste [18]. In the context of the development of this pharmaceutical form, in the present work, we partially changed the composition of the core (agar and sunflower oil were replaced with stevia) while the coating was made with dark chocolate instead of milk. The most important challenge, however, was the attempt to incorporate two active ingredients (DIP and ACA) into the core of the preparation instead of one, of which the second one presents significant instability due to hydrolysis. Of course, it is very important when developing a new generic drug, especially when it is intended for children, to take seriously not only the side effects of the active ingredient but also those of the excipients (e.g., allergies) [19,20].
In general, the approval of such formulations for per-os administration requires the development of a reliable method for the determination of the active ingredients as well as the study of their release and behavior in the gastrointestinal tract.
From an analytical point of view, international literature contains relevant references for the simultaneous determination of dipyridamole and aspirin, using different chromatographic methods (Table 1). In addition to aspirin and dipyridamole, equally important is the determination of salicylic acid (SAL), which is the major impurity resulting either from the synthesis process of acetylsalicylic acid or from its degradation [21].
The aim of the present study was to develop a reliable and sensitive Diode Array Detector/Fluorescence Detector (HPLC-DAD/FLD) method for the quantitative determination of dipyridamole and acetylsalicylic acid in a honey-based formulation with a chocolate coating (15 mg ACA and 75 mg DIP). Since the rapid and easy hydrolysis of ACA to salicylic acid (SAL) is possible, particular attention was given to study the stability of both ACA and DIP in various solvents/buffer solutions as well as in gastric, intestinal fluids, and blood serum (at 37 °C). Subsequently, in the context of simulating the digestion of the new formulation, a three-stage in vitro protocol (oral cavity, gastric, intestinal) was applied to study the release rate of DIP, ACA and SAL [28]. Finally, a fraction of the intestinal fluids was transferred to the donor compartment of Franz cells, in order to investigate the degree of penetration of the active ingredients into the systemic circulation. The information provided could be used as basic knowledge for the study of corresponding modern pediatric formulations.

2. Materials and Methods

2.1. Instruments and Equipment

For the study and quantification of the active pharmaceutical ingredients (APIs), a Shimadzu HPLC arrangement with two LC-20AD pumps, a SIL-20A HT automatic sampler (injection volume: 20 μL) and a CTO-20A column oven (Shimadzu, Kyoto, Japan) was used. The set-up was connected in series with two detectors, an ultraviolet photodiode array (SPD-M20A) and a fluorescence, RF20-A (Shimadzu, Tokyo, Japan). RF20-A was set at Gain: ×4 and at medium sensitivity. The stationary phase was a C18 Supelco Discovery® HS column (250 mm × 4.6 mm, 5 µm) and the mobile was a mixture of two solvents, (A) H2O/Formic acid 0.2% and (B) Acetonitrile-H2O 90:10 v/v, in isocratic elution (65:35 v/v) and flow rate 1 mL/min.

2.2. Reagents and Solvents

The solvents used were of HPLC grade. Acetonitrile (ACN) and methanol (MeOH) were purchased from VWR Chemicals (Radnor, PA, USA), whereas formic acid (FA) was from Sigma Aldrich (St. Louis, MO, USA). Accordingly, water was of high purity (18.2 MΩ·cm resistivity) (Adrona SIA, Riga, Latvia).
The analytes were dipyridamole, DIP, (purity > 98.0%, Sigma Aldrich, St. Louis, MO, USA), acetylsalicylic acid or aspirin, ACA, (purity > 98.0%, TCI, Zwijndrecht, Belgium) and salicylic acid, SAL, (Sodium salicylate, purity > 99.5%, Reagenzien Merck, Darmstadt, Germany). Their chemical structures are presented in Figure S1.
Edible materials were used to produce the pharmaceutical formulation: Greek thyme honey, stevia which is a natural sweetener, preservative approved as safe (GRAS) by the FDA [29,30] and black couverture. All materials were purchased from local stores in Thessaloniki, Greece. For the in vitro permeability experiments, dialysis tubing cellulose membranes, DT9527-100FT, (obtained from Sigma-Aldrich, St. Louis, MO, USA) was utilized.

2.3. Solutions

2.3.1. Stimulated Fluids

Three stimulated digestion fluids (Text S1) were prepared in order to perform the in vitro digestion protocol [31]: one for the oral cavity (Simulated Salivary Fluid, SSF), one for the stomach (Simulated Gastric Fluid, SGF) and one for the intestinal tract (Simulated Intestinal Fluid, SIF). Accordingly, for the permeability study of the new formulation in Franz cells, phosphate-buffered saline (PBS) solution was prepared according to the procedure described in Text S2 [32].

2.3.2. Stock Solutions and Diluent

To prepare the stock solutions of the three APIs, 10.0 mg of each API were accurately weighed into a 25 mL volumetric flask and after adding 1 mL of methanol the samples were sonicated for 1–2 min. Subsequently, they were filled to the mark with acetonitrile and stored at 2 °C. From the stock standard solutions, appropriate dilutions were made to produce two series of six standard solutions (one for the UV and one for the FLD detector). The final diluent was a mixture of water:acetonitrile in a 60:40 v/v ratio.

2.4. Preparation of the Formulation

The proposed formulation was prepared with a specific dosage of active ingredients (APIs) and excipients, so that it can be administered daily (two-three single doses, daily) and to children over 5 years of age. Specifically, a single dose of the formulation contains 75 mg DIP and 15 mg ACA as active ingredients, while 2 g honey, 220 mg stevia and 2 g dark chocolate were used as excipients. In order to carry out the necessary experimental procedures, 20 doses of the suggested preparation were formulated according to the following procedure: 1.5 g of DIP and 300 mg of ACA were accurately weighed and transferred into a glass beaker. Then, liquid stevia (4.4 g) was gradually added, with simultaneous stirring/sonicating and gentle heating in a water bath (35 °C for 2–3 min) until complete homogenization. Then, 40 g of honey was also added with continuous mixing (at 25 °C for 5 min). The final mixture was kept in the refrigerator at 2 °C to fill the chocolate mold, which serves as a coating for the pharmaceutical preparation.
For the coating process, 40 g of couverture chocolate was melted in a water bath (60 °C) and, before the chocolate solidified, poured into 20 silicone molds (2 × 1 cm, wall thickness 1 mm). Then, after being placed in the refrigerator for about 2–3 h to solidify the chocolate, they were filled with the honey core. The flat surface of the preparation was covered with a thin layer of chocolate and placed in the refrigerator for another 3 h (Figure 1).

2.5. Pretreatment of Samples (Preparation) Before Analysis

Each formulation consists of a honey core containing APIs and an outer chocolate coating (Figure S1). For its pretreatment, the chocolate coating was carefully cut and the honey core was quantitatively transferred (rinsed with 15 mL of methanol) into a 25 mL beaker. The sample was placed in an ultrasonic bath for 2.5 min and after removing the remaining coating, it was sonicated for another 2.5 min. Subsequently, 1 mL of the solution was transferred to a 25 mL flask which, after being placed in an ultrasonic bath, was filled, dropwise, with acetonitrile. The sample was sonicated for an additional 20 min, placed in the freezer for 30 min and centrifuged (5000 rpm for 15 min). Finally, 1 mL of the sample was diluted to a final volume of 10 mL (diluent: H2O-ACN 60:40 v/v), filtered (0.45 μm PTFE filter) and analyzed by the proposed HPLC method.

2.6. In Vitro Digestion Protocol

The digestion protocol was tested in three replicates (formulations). Each sample was processed with a chewing simulation device (for two minutes to simulate mastication by a young patient) and after the addition of 5 mL oral solution, it was placed into a shaking water bath, at 37 °C (2 min). Since the first stage was complete, 10 mL of gastric solution was added to the oral bolus. The mixture was left at the same experimental conditions for 2 h (gastric phase). In the final digestion phase, gastric chyme was mixed with 19.85 mL intestinal solution (pH 7) and left for 2 h in the shaking water bath (37 °C). During the study, samples (500 μL) were taken at regular intervals at the 1st and 2nd hour (gastric phase) as well as in 2.5, 3 and 4 h (intestinal phase).

2.6.1. In Vitro Samples Pretreatment

The 500 μL samples were lyophilized (Virtis Advantage Plus device from SP Scientific, Warminster, PA, USA) and then 2 mL of methanol was added to them. Subsequently, the sample was subjected to an ultrasonic bath (5 min) and vortex (2 min) in order to reconstitute the APIs without recovering the salts. A volume of 1 mL of the supernatant solution was transferred into a tube to which 2 mL of ACN was added dropwise (while the sample was subjected to ultrasonication for 20 min) in order to sediment the chocolate and honey components. The sample was then left in the freezer for 30 min, centrifuged (5000 rpm for 15 min), diluted (2 mL of supernatant + 1 mL H2O), filtered (0.45 μm PTFE filter) and analyzed.

2.6.2. Sediment Reconstitution

At the end of the in vitro digestion method, the two APIs (DIP and ACA) and the degradation product (SAL) were determined in the precipitate, according to the following reconstitution procedure. The sample (precipitate and supernatant) was frozen (10 min), centrifuged (4500 rpm for 10 min) and the supernatant was removed (by decantation). Then, 10 mL of MeOH was added to the precipitate and the sample was sonicated for 5 min. In addition, 1 mL of the supernatant was transferred into a 10 mL flask which was then filled dropwise to the mark with acetonitrile, with simultaneous sonication (for 20 min). Then, the sample was placed in the freezer (30 min), centrifuged (15 at 5000 rpm), diluted (1 mL of supernatant + 1.5 mL H2O), filtered and analyzed by HPLC.

2.7. In Vitro Permeability Study

To ensure consistency, the concentration of APIs in the digested intestinal samples was quantified before application to the Franz diffusion cells. Then, after the digestion procedure, 2 mL of intestinal fluids was placed in the donor compartment, whereas the acceptor contained degassed PBS at pH 7.4. The procedure was performed under stirring at 90 rpm (37 °C ± 0.2 °C). At specified time intervals (0.5, 1 and 2 h), 0.5 mL of the samples was taken from the receptor compartment and replaced with an equal volume of freshly prewarmed receptor medium. The concentration of each API was quantified, using the suggested HPLC method (Section 3.1). Blanks, that contained PBS, were pretreated with the same procedure. Each sample was analyzed in triplicate and its cumulative release over time was plotted. The steady-state flux (Jss) was determined by plotting the permeation of API per unit area (μg/cm2) against time (h) and calculating the slope of the linear portion of the resulting line. The following equation was applied to calculate the apparent permeability coefficient (Paap).
Papp = Jss/Cd (cm/h),
where Cd is the initial concentration of the drug in the donor compartment and Jss is the steady state flux.

3. Results and Discussion

3.1. Chromatographic Method Development

The purpose of the following investigation was to suggest a reliable analytical method that would meet the chromatographic requirements, i.e., satisfactory peak shape (Tf = 1 ± 0.2), effective separation (Rs ≥ 1.5) and short analysis time (tR < 20 min). In addition to the two APIs (ACA and DIP), special emphasis was also given to the adequate separation of salicylic acid (SAL), which is a degradation product of ACA and is considered an indicator of its hydrolysis.
In order to select the optimal stationary phase, different types of reversed-phase columns (phenyl, C8, C18, -CN and -NH2) were tested, under similar analytical conditions. In Table S1, a summary of all the experimental investigations is presented. In general, the main problem that had to be addressed was the insufficient separation of ACA and SAL, which mainly co-elute immediately after the solvent front, while the DIP peak was usually delayed. Reducing the organic–mobile phase ratio, increased the elution time of both peaks but did not separate them sufficiently, while DIP was delayed further. In most cases, also, the acidic pH of the mobile phase gave a triple peak in SAL, broken and with the solvent front, while ACA was wide and broken. Comparatively better behavior was observed in C18 type columns, of different dimensions (150 and 250 mm). Of these, Supelco Discovery® HS (25 cm × 4.6 mm, 5 μm) provided the best separations, with sharp peaks, short analysis time and isocratic elution.
Acetonitrile was used as the organic solvent in the mobile phase instead of methanol mainly because it improved the peak shape for DIP (Tf MeOH = 1.5 and TfACN = 1.05) and gave low backpressure. Another notable observation was that very small changes in the concentration of the organic solvent (ACN) in the mobile phase significantly affected the retention time of DIP and its elution order, compared to ACA and SAL (Figure 2). The optimum mobile phase was a mixture of 65:35 v/v of two phases (A): aqueous solution at pH 2.4 with H2O and Formic acid 0.2%, and (B) acetonitrile-H2O 90:10 v/v, in isocratic elution.
To select the optimal pH value of the mobile phase and in order to prevent hydrolysis of ACA, it had to be acidic [33]. Different acids such as formic, acetic or trifluoroacetic as well as phosphate buffers were tested. Based on the pKa values of the analytes (Table S2) [34,35,36,37,38,39,40,41,42,43,44,45] and using phosphate buffer, three pH (2.8, 3.5 and 4.25) were tested. The two APIs, which are acids (pKa(SAL) 2.98 and pKa(ACA) 3.5) at pH values higher than their pKa, are ionized and their retention time is reduced. DIP, on the other hand, as a base (pKa 3.54), at pH > pKa, due to its non-ionization, is eluted at tr > 20 min. Therefore, lower pH values were considered more appropriate since they delay the elution of the two acids and accelerate that of dipyridamole (20 mM NaH2PO4 at pH = 2.8 with phosphoric acid).
Compared to phosphate buffer and the other acidifying agents, formic acid gave the best chromatogram (Table 2) and was chosen for pH adjustment. Specifically, phosphate showed more noise in the baseline, while the other two acids (formic or trifluoroacetic) gave worse tailing factor values (Tf > 1.1) for DIP. Formic acid was added only to the aqueous phase, as it did not improve the result when added to the organic phase. It was studied at three concentration levels (0.05%, 0.1%, and 0.2%) and 0.2% was selected as optimal due to the sharpest peaks.
The choice of the appropriate diluent in the final samples was based on the solubility of the APIs, their stability (especially of ACA) and the resulting chromatograms. According to the stability study (Section 3.3) the solvent that keeps ACA stable, for at least 8 h, was acetonitrile or a mixture of ACN-H2O of 30:70 v/v. Because 100% acetonitrile affects the quality of the chromatogram (broken and broadened DIP peak), a 60:40 v/v H2O-ACN mixture was preferred (Figure 3).
An ultraviolet detector was utilized for the quantification of DIP, SAL and ACA in the pharmaceutical formulation. Based on the literature and the obtained UV spectra (Figure S2), acetylsalicylic acid exhibits a maximum absorbance at 230 nm, salicylic acid at 237 nm and dipyridamole at 285 nm.
The same detector and λmax values were used for SAL and ACA in both digestion and permeability experiments. However, for DIP, an additional peak (contamination), with a retention time similar to that of the analyte, appeared in the chromatogram of the digestion substrate samples. In order to eliminate this interference and since it does not absorb at λ = 405 nm, the determination of DIP was conducted at this wavelength. Respectively, for the permeability study, in order to detect DIP at very low concentration levels, a fluorescence detector (set at λexc = 285 nm and λem 480 nm) was used. The two other analytes (ACA and SAL) were not measured by the fluorescence detector because they did not provide reliable results (%RSD > 5.5, r2 < 0.840).

3.2. Method Validation

3.2.1. System Suitability

A crucial part of the evaluation of an analytical method is the system suitability control. The suitability control parameters depend on the procedure being validated and must meet the specified requirements (Table 2). The validation of the proposed analytical method was carried out based on the International Conference on Harmonization of Technical Requirements for the Registration of Medicinal Products for Human Use (ICH Q2) guidelines [46].

3.2.2. Selectivity

The selectivity of an analytical method is defined as its ability to identify the compound of interest in the presence of other contaminations in the sample (impurities, degradation products, substrate components, etc.). In practice, this means that in the chromatograms the retention times of the analytes and the possible contaminations should not be identical. Therefore, a comparison between two blanks (with diluent ACN-H2O 40:60 v/v and honey extracts) and two standard solutions (with DIP, ACA and/or SAL) was made (Figure 4).

3.2.3. Linearity

The linearity of the chromatographic method was investigated at six different concentrations. During the analysis of the samples, three replicates were performed for each concentration level. The concentration vs. absorbance data were correlated with the linear regression analysis (Table 3). The good linearity of the method was also estimated by the % y-intercept values (intercept value × 100/100% response) for each analyte which should be <2%.

3.2.4. Precision Repeatability

To validate the analytical method, a series of measurements were performed by the same analyst in a short period of time on the same HPLC instrument. The parameters examined were within-day precision (repeatability) and between-day precision (intermediate precision) over three consecutive days. Repeatability was checked over the entire concentration range, at three levels: the lowest, the medium and the highest. For the lowest and the highest concentrations, four repetitions were performed, while for the medium six. For the intermediate precision, the average of the repetitions was calculated, at the three concentration levels between three days. The repeatability results were within the acceptable limits (Table 4).

3.2.5. Accuracy

For the determination of accuracy, six standard solutions of the three analytes were prepared and analyzed at six concentration levels. The equations from the calibration curves were used to calculate the actual values of their concentrations and their % recovery which ranged from 99.9–100.8% for DIP, 95.4–101.1% for ACA and 99.5–101.0% for SAL (using DAD as a detector) and 98.2–101.0% for DIP by the FLD detector (Table S3).

3.2.6. Limit of Detection (LOD) and Quantification (LOQ)

The limit of detection and quantification of the three analytes were calculated (Table 2) according to the following Equations (1) and (2):
LOD = 3.3 × SD/Slope
LOQ = 10 × SD/Slope
where SD is the standard deviation of the intercept of the calibration curve.

3.2.7. Robustness

To evaluate the robustness of the chromatographic system, the effect of small changes (±1) on its operating parameters at the tailing factor (Tf) and the AUC value (area under the curve) were investigated for each API separately (Table 5).
According to the results, the system is robust to small changes in λmax (nm) and temperature, while the mobile phase flow rate significantly affects the AUC values (areas under the curve).

3.3. Stability Study of the Analytes

The hydrolysis of acetylsalicylic acid into salicylic and acetic acid is a phenomenon that had to be taken seriously into account in all analytical and preparative processes of the suggested formulation (honey/chocolate). In contrast, DIP generally exhibits stability.
According to a recent study carried out with the aim of finding the solvent that ensures the greatest stability of ACA, acetonitrile and 1,4-dioxane were suggested, while methanol and ethanol were shown to create the greatest instability [47]. The degradation of ACA, in substituted and unsubstituted polyhydric alcohols, in contrast to its stability in acetonitrile with 1% formic acid solution, has also been recorded by other researchers [48,49]. Accordingly, in aqueous solutions, ACA exhibits stability in a mixture with polyethylene glycol, while it is largely hydrolyzed in phosphate buffer at pH 7.0 and 7.4 and a temperature of 37 °C [33]. Degradation studies have also been performed on mixtures of water with propylene glycol and triethylene glycol diacetate with ethanol, diethyl ether and diethylene glycol (at various concentrations and temperatures with or without sonication) [50,51]. Considering these reports, it was considered appropriate to conduct a further stability investigation of ACA and the two other analytes, SAL and DIP.

3.3.1. Stability in Different Solvents

The stability test was carried out for a period of approximately 8 h, which is usually the duration of each analysis, and the following diluents were tested: acetonitrile (ACN), acetonitrile-H2O 30:70 v/v, methanol (MeOH), methanol-H2O 50:50 v/v, H2O with F.A 0.2%, NaOH 0.1 N. According to the results presented in Figure 5, DIP generally exhibits stability in all solvents while ACA does not. The decomposition of ACA to SAL in 0.1 N NaOH solution is complete and instantaneous, while its instability in methanol or methanolic solutions is characteristic. Finally, it is noteworthy that whatever amount of % ACA is hydrolyzed is converted equivalently to SAL.
Considering the stability study of the two substances as well as their solubility properties (Table S2), their stock solutions were prepared as follows: Initially, 1 mL of methanol was added, the sample was sonicated (5 min) and then made up to 25 mL with ACN. Acetonitrile can also be used as a diluent for the intermediate standard solutions, in which the APIs are stable even under ultrasonic conditions (30 min). Accordingly, the 40:60 v/v acetonitrile–water mixture was chosen as the optimal diluent of the final standard solutions, providing stability and optimal chromatograms (Figure 2).

3.3.2. Stability at 37 °C in Different Medias

In order to study the release of the two APIs, dipyridamole and acetylsalicylic acid or its hydrolysis product, salicylic acid, into the gastrointestinal tract, two types of digestive fluids were used: Simulated Gastric (SGF) and Simulated Intestinal Fluid (SIF). In addition, PBS was used to study their permeability in Franz cells. Therefore, a stability study (within 3 h) of the three analytes at the three media (37 °C) followed to identify their degradation.
Based on the graphs depicted in Figure 6, their relative stability was higher than 80%. The greatest losses occur, as expected, at acetylsalicylic acid in SIF and PBS, as these solutions are alkaline.

3.4. Formulation Studies

3.4.1. Preparation Processing Method

For the design of the liquid–solid extraction process, the appropriate solvent had to be selected for the quantitative and selective recovery of the active ingredients from the substrate (honey with or without chocolate coating). Based on their solubility properties (Table S2), water cannot be used as such, because on the one hand, it does not ensure the complete dissolution of the active ingredients, and on the other, it dissolves most of the components of the substrate (honey and chocolate) [52]. Therefore, two solvents were investigated, methanol and acetonitrile, of which methanol exhibits the greatest solvating capacity for both active ingredients but does not ensure the stability of ACA. Similarly, to investigate which of them does not dissolve honey, 500 mg of the substrate was weighed into two separate beakers, one of which was filled with 25 mL methanol and the other with acetonitrile. The two solutions were sonicated (15 min) and then placed in the freezer (30 min). According to Figure S3, methanol, unlike acetonitrile, does not show selectivity, since it completely dissolves honey.
Therefore, methanol should, of course, be used in the extraction (as the optimal solvent of ACA, DIP) but in the minimum possible amount in order not to dissolve the substrate. Also, the sample, at this stage, should not be subjected to sonication for a long time in order not to destroy the ACA. Then, after the first dilution, the ratio of acetonitrile to methanol should be as high as possible to precipitate the honey. Various MOH-ACN ratios were tested (Table S4) and 1:24 v/v was considered optimal. The sample was then sonicated (30 min), frozen (30 min), centrifuged (5000 rpm for 15 min), diluted (1 mL in 10 mL of 60:40 v/v H2O-ACN diluent) and filtered before analysis by HPLC.
The proposed method was applied to five chocolate–honey samples and the % recovery of the substances was calculated. According to the results, the % recoveries were found to be 97% (%RSD = 1.82) for dipyridamole and 100.1% (%RSD = 1.78) for acetylsalicylic acid.

3.4.2. Formulation Stability Study

A short-term stability study of the formulation was carried out to evaluate the compatibility and preservation of the substances in the honey substrate. More specifically, the two active ingredients, ACA and DIP, were quantitatively determined in the formulation which was stored at 2 °C for 0, 1, 2, 5, and 7 days. Up to the 7th day, the recovery values of both active ingredients were still >95%.

3.4.3. Drug Release and Permeability Assessment

Referring to the literature data, dipyridamole is a poorly water-soluble drug, and its degree of dissolution decreases with pH increase. It is absorbed better in the small intestine [53]. On the other hand, aspirin, which is widely co-administered with dipyridamole [9], seems to be absorbed both in the intestinal epithelium (its ionized form) [54] and the stomach (non-ionized form) [55]. Of course, for its study, its significant rate of hydrolysis into salicylic acid (under alkaline conditions) should not be overlooked.
Under the present circumstances, it was of interest to study the behavior of ACA and DIP in the gastrointestinal (GI) tract when incorporated into the chocolate/honey composition. Therefore, the proposed formulation was subjected to simulated gastrointestinal digestion using a standardized in vitro procedure [28]. For the study of the two active ingredients, the significant hydrolysis rate of ACA to salicylic acid (under alkaline conditions) also had to be taken seriously.
Another point that should be clarified is that the in vitro standardized digestion model used in this study is based on adult gastrointestinal physiology. It is therefore important to recognize the limitations this imposes when predicting in vivo behavior in pediatric populations, due to physiological differences [56]. For example, children may exhibit slower gastric emptying times, lower enzyme activity, reduced bile salt concentration, and a different pH profile throughout the gastrointestinal tract [57,58]. These factors can significantly affect the solubility, stability, and release kinetics of orally administered drugs. As a result, drug behavior observed under adult-simulating conditions may not fully represent the dynamic digestive environment of younger children. However, because the present formulation is primarily intended for older pediatric patients or adults, it was studied in the standard digestive model. According to the model results, the behavior of both substances in the GI tract depends on their physicochemical characteristics. Specifically, the release of the drugs from the chocolate/honey formulation, which differs significantly between the two APIs, highlights the significant influence of the medium (gastrointestinal fluids) on the kinetics of both drugs’ dissolution. In this case and in order to consider the hydrolysis product of ACA, SAL, its release rate was calculated overall, summing both substances. Dipyridamole (weak base) is a poorly water-soluble substance that exhibits enhanced dissolution in acidic gastric conditions (Figure 7a), resulting in its supersaturation and then, during passage into the intestinal environment, its partial precipitation [59]. Notably, the presence of lipids and digestive enzymes, during digestion, has been shown to significantly enhance the solubility and supersaturation of weakly basic drugs, thereby affecting their release [60]. In contrast, aspirin, being a weak acid, displays poor solubility in acidic gastric conditions, potentially leading to precipitation in the stomach and associated gastrointestinal side effects [61]. As the environmental pH increases towards intestinal conditions, aspirin’s solubility significantly improves, resulting in the notable increase in its release observed during the intestinal phase of the in vitro digestion experiment (Figure 7a).
Following in vitro digestion, permeability studies through cellulose membranes were performed to further evaluate the permeation potential of both drugs. Franz diffusion cells are commonly employed to study passive drug permeability, including applications in oral formulations [62]. Although the arrangement does not mimic active transport or enzymatic degradation processes of the intestinal epithelium, it offers a standardized method to evaluate drug diffusion post-digestion. The present study was used to compare the permeation potential of ACA and DIP after simulated gastrointestinal digestion. Notably, Klitgaard et al. [63] employed a similar approach, transferring digested samples from in vitro lipolysis to Franz cells to assess drug permeability, supporting the relevance of this method in evaluating post-digestion drug transport.
As shown in Figure 7b and Table 6, dipyridamole exhibited limited permeation behavior (Papp = 0.1178 ± 0.0121) due to its low solubility in alkaline conditions. In contrast, ACA + SAL (Papp = 0.3233 ± 0.1219) rapidly achieved a high permeation level, indicating stable and efficient permeation across the membrane. (Figure 7b). The observed permeability profiles suggest that ACA may achieve faster, and more efficient absorption in vivo compared to DIP. This inference aligns with the Biopharmaceutics Classification System (BCS), where ACA is classified as a Class I compound (high solubility, high permeability) [40], and DIP falls under Class II (low solubility, high permeability [64]. The higher permeability of ACA observed in vitro correlates with its known rapid absorption and onset of action in clinical settings [65]. To bridge the in vitro findings with in vivo performance, it is essential to consider studies that have established in vitro–in vivo correlations (IVIVC). For instance, Klitgaard et al. [63] demonstrated a linear IVIVC for self-nanoemulsifying drug delivery systems (SNEDDS) by comparing the area under the curve (AUC) from in vitro permeation studies with in vivo plasma concentration profiles. Their findings underscore the potential of in vitro models, to predict in vivo pharmacokinetics for certain formulations. Based on our in vitro data and supported by the aforementioned studies, we anticipate that ACA will exhibit rapid absorption and onset of action in vivo, consistent with its clinical profile. DIP, while demonstrating moderate permeability in vitro, may benefit from formulation strategies aimed at enhancing its solubility and, consequently, its bioavailability. Nevertheless, we acknowledge that in vitro models cannot fully replicate the complexities of human physiology. Factors such as first-pass metabolism, enzymatic degradation and individual patient variability can influence the actual in vivo pharmacokinetics. Therefore, while our in vitro findings provide valuable insights, subsequent in vivo studies are necessary to validate these predictions and fully characterize the pharmacokinetic profiles of DIP and ACA.
A particularly noteworthy observation in the overall release process (digestion and penetration stage) of the two active substances is related to the hydrolysis rate of ACA to SAL. According to the bar graph in Figure 8, while in the gastric phase, ACA is already hydrolyzed to a rate of >60%, this rate increases (>80%) in the intestinal tract. Then, during the study of the penetration rate of both substances, in Franz cells, approximately the same amounts of ACA and SAL were detected at the acceptor compartment (despite the low content of ACA in the intestinal tract). This leads to the conclusion that the permeation rate of ACA is much higher than that of SAL (Figure 8) and is due to their different physicochemical properties. It is noteworthy that, while both compounds show increased solubility at higher pH due to ionization, their permeation is also affected by their partition coefficients and molecular interactions with the membrane. The log p value of SAL (2.26) is higher than that of ACA (1.19), reflecting SAL’s greater lipophilicity due to its phenolic structure [66]. However, in the context of Franz diffusion cells using hydrophilic cellulose membranes, the less lipophilic ACA may achieve better partitioning and diffusion across the membrane interface, favoring its permeation.
Finally, it should be clarified that the release kinetics of the proposed chocolate–honey formulation differ significantly from that of the commercial product Aggrenox®. Aggrenox® is designed to provide sustained antiplatelet activity [9] while the proposed formulation is an immediate release (chocolate/honey) matrix. Given these differences, a direct kinetic comparison is not indicated. However, our formulation may offer an alternative for pediatric or dysphagic patients requiring short-term antiplatelet therapy, especially when long-term maintenance therapy with capsule formulations is not feasible.

4. Conclusions

Considering the bitter taste of most APIs upon ingestion, a palatable formulation consisting of a honey core and a dark chocolate coating was created. Two active ingredients, dipyridamole and acetylsalicylic acid, were successfully incorporated into the core and the new formulation was quantified using a validated reverse-phase HPLC method with UV and FLD detectors. For the suitability of the proposed dosage formulation, stability studies of the active ingredients, their release in the gastrointestinal tract (oral cavity, stomach and intestine) and an in vitro study of their permeation with Franz cells were followed. The present research could be considered a first step toward the preparation of modern and easy-to-consume formulations, using edible materials as excipients. Additional optimization, bioequivalence, and stability studies are required for their safe future use.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15116048/s1, Figure S1. Chemical structure of: (a) dipyridamole, (b) acetylsalicylic acid and (c) salicylic acid; Text S1. Digestion Fluids Solutions; Text S2. Phosphate Buffer Saline Solution; Table S1. Stationary phase investigation; Table S2. Summary table of physicochemical properties of the three APIs; Table S3. % Recovery of three analytes for: (a) UV and (b) Fluorescence detector; Table S4. % Recovery of DIP and ACA in different solvents after exposure to ultrasonic for 30 min Figure S2. UV spectra of the analytes; Figure S3. Honey solution at Methanol (left) and Acetonitrile (right).

Author Contributions

Conceptualization, C.K.M.; methodology, G.K., N.P., K.C. and C.K.M.; validation, G.K. and N.P.; formal analysis, G.K., N.P. and K.C.; writing—original draft preparation, C.K.M.; writing—review and editing, G.K., N.P. and C.K.M.; visualization, G.K.; supervision, C.K.M.; project administration C.K.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chocolate formulation with honey core.
Figure 1. Chocolate formulation with honey core.
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Figure 2. Effect of acetonitrile on the retention time (tR) of the analytes. Six different ratios were tested, three times each, in terms of reproducibility.
Figure 2. Effect of acetonitrile on the retention time (tR) of the analytes. Six different ratios were tested, three times each, in terms of reproducibility.
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Figure 3. Chromatograms used as diluent: (a) H2O/MeOH 50/50 (b) MeOH 100% (c) ACN/MeOH 50/50 (d) H2O 100% (e) H2O/ACN 60/40 (f) ACN 100%.
Figure 3. Chromatograms used as diluent: (a) H2O/MeOH 50/50 (b) MeOH 100% (c) ACN/MeOH 50/50 (d) H2O 100% (e) H2O/ACN 60/40 (f) ACN 100%.
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Figure 4. Chromatograms of (a) a standard solution of DIP, ACA and SAL, (b) a standard solution of DIP and ACA, (c) a sample obtained from the honey–core formulation, (d) blank solvent, (e) blank honey solution.
Figure 4. Chromatograms of (a) a standard solution of DIP, ACA and SAL, (b) a standard solution of DIP and ACA, (c) a sample obtained from the honey–core formulation, (d) blank solvent, (e) blank honey solution.
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Figure 5. Stability of acetylsalicylic acid, salicylic acid and dipyridamole at: ACN (dark blue), ACN/H2O-30/70 (red), MeOH (green), MeOH/H2O-50/50 (purple), H2O/FA 0.2% (pale blue), 0.1 N NaOH (orange), n = 3 and RSD < 1.5%.
Figure 5. Stability of acetylsalicylic acid, salicylic acid and dipyridamole at: ACN (dark blue), ACN/H2O-30/70 (red), MeOH (green), MeOH/H2O-50/50 (purple), H2O/FA 0.2% (pale blue), 0.1 N NaOH (orange), n = 3 and RSD < 1.5%.
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Figure 6. Stability study of DIP (blue), ACA (red), SAL (green) in simulant fluid SGF, SIF and PBS at 37 °C, n = 3 and RSD < 1.5%.
Figure 6. Stability study of DIP (blue), ACA (red), SAL (green) in simulant fluid SGF, SIF and PBS at 37 °C, n = 3 and RSD < 1.5%.
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Figure 7. In vitro release (a) and permeability (b) profiles of dipyridamole and aspirin-salicylic acid encapsulated in chocolate-based formulations. Values represent mean ± SD (n = 3).
Figure 7. In vitro release (a) and permeability (b) profiles of dipyridamole and aspirin-salicylic acid encapsulated in chocolate-based formulations. Values represent mean ± SD (n = 3).
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Applsci 15 06048 g008
Figure 8. ACA (dark grey) hydrolysis rate in SAL (pale grey) at gastric, intestinal and phosphate-buffered saline.
Figure 8. ACA (dark grey) hydrolysis rate in SAL (pale grey) at gastric, intestinal and phosphate-buffered saline.
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Table 1. Methods for the simultaneous determination of ACA, DIP, and SAL/literature review.
Table 1. Methods for the simultaneous determination of ACA, DIP, and SAL/literature review.
Reference TitleComments
Development of RP-HPLC method for simultaneous evaluation of uniformity of dosage units from aspirin and dipyridamole extended-release capsules [22]HPLC PDA/UV-Vis
Stationary phase: X Bridge C8 column (250 × 4.6 mm, 5 μm)
Mobile phase: 0.05 Μ phosphate buffer pH 2.5-MeOH 55:45 v/v
LOD: ACA 0.56 µg/mL, DIP 3.95 µg/mL
LOQ: ACA 1.69 µg/mL, DIP 11.96 µg/mL
Simultaneous Determination of aspirin, Dipyridamole and Two of Their Related Impurities in Capsules by Validated TLC-Densitometric and HPLC Methods [21]TLC-Densitometric
Diluents: MeOH
LOD: SAL 0.26 µg/band
LOQ: SAL 1.0 µg/band
HPLC-UV
Stationary phase: Zorbax ODS (5 µm, 250 mm × 4.6 mm i.d.)
Mobile phase: phosphate buffer pH 3.3: ACN: triethylamine (40:60:0.03)
LOD: SAL 0.70 µg/mL
LOQ: SAL 2.5 µg/mL
Rapid and simultaneous determination of aspirin and dipyridamole in pharmaceutical formulations by RP-HPLC method [23]Detector: PDA
Stationary phase: Waters Symmetry C18 (3.5 µm, 50 × 4.6 mm)
Mobile phase: 0.1% o-H3PO4: ACN (75:25)
Concentration range: ACA (0.5–10 µg/mL), DIP (4–80 µg/mL)
Development and validation of a rapid method RP-UPLC for the determination of aspirin and dipyridamole in combined capsule formulation [24]Stationary phase: Hypersil Gold C18 (1.9 µm, 100 mm × 2.1 mm)
Mobile phase: phosphate buffer and triethylamine, pH 2.5- MeOH 50:50 v/v
Stability-Indicating Spectrofluorimetric and RP-HPLC Methods for the Determination of Aspirine and Dipyridamole in their Combination [25]Detector: FLD
Diluents: CH3COOH in CHCl3, 1% v/v
LOD: ACA 0.21 µg/mL, DIP 0.37 µg/mL, SAL 0.25 µg/mL
LOQ: ACA 0.97 µg/mL, DIP 1.28 µg/mL, SAL 0.36 µg/mL
RP-HPLC- UV
Stationary phase: Adsorbosil C8 (10 μm, 250 mm × 4.6 mm i.d.)
Mobile phase: H2O- ACN: o-H3PO4 (65:35:2, v/v/v)
LOD: ACA 0.026 µg/mL, DIP 0.044 µg/mL, SAL 0.015 µg/mL
LOQ: ACA 0.074 µg/mL, DIP 0.153 µg/mL, SAL 0.085 µg/mL
Spectrofluorometric estimation of aspirin and dipyridamole in pure mixtures and in dosage forms [26]Diluents: CH3COOH in CHCl3, 1% v/v
Concentration range: ACA 2–12 µg/mL, DIP 2–12 µg/mL
Simultaneous Determination of Dipyridamole and Acetylsalicylic Acid in Pharmaceuticals and Biological Fluids by Synchronous and First Derivative Synchronous Fluorimetry [27]Diluents: MeOH, phosphate buffer pH 7.4
Concentration range: ACA 5–100 ng/mL, DIP 5–90 ng/mL
LOD: 0.05–1 ng/mL
Table 2. System suitability parameters of the proposed method.
Table 2. System suitability parameters of the proposed method.
AnalytesRetention Time (tr)Tailing Factor (Tf)Capacity (k’)Resolution (Rs)Number of Theoretical Plates (N)HETP * × 103 USP
DIP5.51.051.375-2265110
ACA7.20.9752.13.6913,81818
SAL10.20.9893.388814,88317
* Height equivalent of a theoretical plate.
Table 3. Linear regression analysis data.
Table 3. Linear regression analysis data.
AnalytesConcentration (μg/mL) *Equation *%y Intercept(R2)LOD (μg/mL)LOQ (μg/mL)
DIP (UV 285 nm)1.6–40y = 67,764.0 ± 104.0 x − 2210.6 ± 2050.80.0810.0990.302
DIP (FLD λexc/λem:285/480 nm)52.3–156.8 *y = 4353.8 ± 67.2 x + 11,278.5 ± 7027.0 *1.610.9990.0110.033
ACA (UV 230 nm) 1.6–40y = 38,234.3 ± 197.6 x + 7042.4 ± 3896.70.460.9990.3361.019
DIP (UV 405 nm)1.6–40y = 15,398.3 ± 57.5 x − 2565.1 ± 1134.70.420.9990.2430.736
SAL (SAL 237 nm)1.6–40y = 50,203.2 ± 71.2 x + 292.1 ± 1403.90.01410.0920.279
* Concentration expressed in ng/mL for DIP with FLD detector.
Table 4. Repeatability and intermediate precision analysis.
Table 4. Repeatability and intermediate precision analysis.
APIsRepeatabilityIntermediate Precision
Concentration (μg/mL)RSD%Concentration (μg/mL)1st Day2nd Day3rd DayRSD%
DIP (UV)1.6 (n = 4)0.541.6 (n = 4)0.540.170.190.43
16 (n = 6)0.2516 (n = 6)0.250.230.110.20
40 (n = 4)0.2540 (n = 4)0.250.781.531.75
DIP (FLD)0.052 (n = 4)0.610.052 (n = 4)0.611.401.611.88
0.104 (n = 6)0.970.104 (n = 6)0.970.991.841.85
0.157 (n = 4)0.700.157 (n = 4)0.700.210.330.53
ACA (UV)1.6 (n = 4)0.861.6 (n = 4)0.861.001.641.63
16 (n = 6)0.2816 (n = 6)0.280.200.391.26
40 (n = 4)0.3940 (n = 4)0.390.881.442.29
SAL (UV)1.6 (n = 4)0.721.6 (n = 4)0.720.841.141.78
16 (n = 6)0.5816 (n = 6)0.580.170.070.91
40 (n = 4)0.3040 (n = 4)0.300.801.551.25
Table 5. Robustness test of the proposed analytical method.
Table 5. Robustness test of the proposed analytical method.
Parameters%RSD
DIPACASAL
AUCTfAUCTfAUCTf
Flow rate mL/min
(0.9, 1.0, 1.1)		9.462.009.770.669.880.71
Temperature °C (29, 30, 31)0.292.050.850.610.181.36
Mobile phase A:Β
(66:34, 65:35, 64:36 v/v)		0.519.870.480.410.380.35
λmax (nm)
(284, 285, 286 και 229, 230, 231 και 236, 237, 238)		0.2650.0480.370.10.820.058
Table 6. Steady state flux (Jss) and apparent permeability values (Papp) of dipyridamole and aspirin-salicylic acid across cellulose membrane.
Table 6. Steady state flux (Jss) and apparent permeability values (Papp) of dipyridamole and aspirin-salicylic acid across cellulose membrane.
APIsJss (μg cm−2 min−1)Papp (cm/min) * 10−2Statistical Significance vs. DIP
DIP0.0769 ± 0.00680.1178 ± 0.0121-
ACA + SAL0.4778 ± 0.15610.3233 ± 0.1219Significant (p = 0.0439)
* Statistically significant difference at p < 0.05 (unpaired t-test, n = 3 per group).
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Kamaris, G.; Pantoudi, N.; Chachlioutaki, K.; Markopoulou, C.K. Analysis of Aspirin and Dipyridamole in a Modern Pharmaceutical Formulation-Drug Release Study and Permeability Assessment. Appl. Sci. 2025, 15, 6048. https://doi.org/10.3390/app15116048

AMA Style

Kamaris G, Pantoudi N, Chachlioutaki K, Markopoulou CK. Analysis of Aspirin and Dipyridamole in a Modern Pharmaceutical Formulation-Drug Release Study and Permeability Assessment. Applied Sciences. 2025; 15(11):6048. https://doi.org/10.3390/app15116048

Chicago/Turabian Style

Kamaris, Georgios, Nikoletta Pantoudi, Konstantina Chachlioutaki, and Catherine K. Markopoulou. 2025. "Analysis of Aspirin and Dipyridamole in a Modern Pharmaceutical Formulation-Drug Release Study and Permeability Assessment" Applied Sciences 15, no. 11: 6048. https://doi.org/10.3390/app15116048

APA Style

Kamaris, G., Pantoudi, N., Chachlioutaki, K., & Markopoulou, C. K. (2025). Analysis of Aspirin and Dipyridamole in a Modern Pharmaceutical Formulation-Drug Release Study and Permeability Assessment. Applied Sciences, 15(11), 6048. https://doi.org/10.3390/app15116048

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