Development of a Rapid LC-MS/MS Method for Simultaneous Quantification of Donepezil and Tadalafil in Rat Plasma: Its Application in a Pharmacokinetic Interaction Study after Oral Administration in Rats

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats.


Introduction
Alzheimer's disease (AD) is a common geriatric illness characterized by neurodegeneration associated with a reduction in cholinergic system activity in the cerebral cortex and other areas of the brain [1]. The cholinergic system plays crucial roles in physiological processes, such as attention, memory, sensory information, learning, stress response, wakefulness, and sleep. Acetylcholine (ACh) is a chemical messenger, neurotransmitter, and neuromodulator that participates in cholinergic system signaling and regulation. As cholinergic and adrenergic signaling is dysregulated owing to substantially reduced ACh synthesis in patients with AD, cholinesterase (ChE) inhibitors are prescribed as the first-line agents for AD management to increase ACh concentration by inhibiting hydrolyzing enzymes, such as acetylcholinesterase (AChE) and butyrylcholinesterase. Donepezil (donepezil hydrochloride, E-2020, arycept™, Eisai, DPZ) is the first selective AChE inhibitor approved for AD management [2]. Molecules 2023, 28, x FOR PEER REVIEW 3 of 15

Chromatographic Conditions
The chromatographic conditions were applied to induce ideal separation and resolution among DPZ, TAD, and LPZ (IS). Various reversed-phase columns, mobile phase com-

Chromatographic Conditions
The chromatographic conditions were applied to induce ideal separation and resolution among DPZ, TAD, and LPZ (IS). Various reversed-phase columns, mobile phase compositions of isocratic and gradient shapes, and flow rates were tested to optimize the separation conditions for the target analytes and IS. In our present study, Phenomenex Luna C18 (50 × 2.0 mm, 3 µm), Phenomenex Kinetex C18 (100 × 2.1 mm, 2.6 µm), and Halo C18 (50 × 2.1 mm, 2.7 µm) columns were tested to determine optimal separation conditions. Among the various columns tested for the analysis, Phenomenex Kinetex C18 (100 × 2.1 mm, 2.6 µm) provided the most appropriate separation of the matrix, high sensitivity, peak shape, and stable reproducibility, whereas other columns had low sensitivity, poor separation, peak tailing, or fronting. Isocratic elution was first utilized to separate the analytes; however, it was difficult to separate the peaks of the analyte and IS owing to the lack of power of the isocratic mobile phase. Therefore, gradient elution was selected to achieve optimal separation and good resolution. As LPZ has been reported to be unstable at low pH [33,34], we designed solvent A with 2 mM ammonium acetate to improve the stability of LPZ and solvent B with 0.1% formic acid in acetonitrile to optimize the peak shape. Acetonitrile was selected as a modifier because it exhibits a higher dissolution capacity from the matrix peak and efficient peak intensity compared to other organic solvents.

Plasma Sample Preparation
DPZ is mostly extracted by using the liquid-liquid extraction (LLE) method using methyl tert-butyl ether, ethyl acetate, and n-hexane as the extraction solvents [27,[35][36][37]. However, TAD is usually extracted using the solid phase extraction and LLE methods using dimethyl ether and dichloromethane as the extraction solvents [17][18][19][20][21]. For simultaneous extraction of DPZ, TAD, and IS with different chemical structural properties, protein precipitation was selected as an extraction method because it is a simple sample preparation procedure with excellent reproducibility [38][39][40]. Acetonitrile and methanol were tested as the precipitants, and a 6.7:1 ratio of acetonitrile and plasma consistently yielded high recoveries with symmetric peak shapes for DPZ, TAD, and IS without matrix interference when compared to those achieved with methanol. Thus, acetonitrile was selected as the precipitant for this experiment.

Specificity and Lower Limit of Quantitation
To verify the selectivity, we utilized six different blank plasmas and pooled the blank plasma at each step. Figure 2 represents the chromatograms of (A) blank rat plasma; (B) LPZ (IS) (20 ng/mL) spiked blank plasma; (C) DPZ (200 ng/mL) spiked blank plasma; (D) TAD (200 ng/mL) spiked blank plasma; (E,F) blank plasma spiked with DPZ (1 ng/mL), TAD (1 ng/mL), and IS (20 ng/mL); and (G) plasma sample from 1 h after oral administration of DPZ (3 mg/kg) and TAD (0.3 mg/kg) with additional IS. During the retention time, the matrix peaks of DPZ (t = 1.32 min), TAD (t = 2.57 min), and IS (t = 2.37 min) did not interfere with each other. The LLOQ of DPZ (1 ng/mL) and TAD (1 ng/mL) were determined with signal-to-noise (S/N) ratios of >10, suggesting a sufficiently sensitive method to quantitate DPZ and TAD in plasma after the oral administration of DPZ and TAD to rats. The precision and accuracy of the LLOQ samples were ensured for the PK study.

Linearity
Using linear regression, calibration curves for DPZ and TAD were plotted with seven concentrations over the range of 1-200 ng/mL. The equation for the calibration curves (n = 5) with the mean ± SD of the slope and the intercept was y = 0.277 (± 0.027) x + 0.033 (±0.007) (r 2 ≥ 0.9904) and y = 0.0402 (± 0.0027) x + 0.001 (±0.003) (r 2 ≥ 0.9966) for DNZ and TAD, respectively. The average slope and intercept of the regression equations were 0.2770 (co-efficient of variation (CV): 8.6%) for DPZ and 0.0403 (CV: 6.0%) for TAD. The linearity was satisfactory and reproducible over time.

Linearity
Using linear regression, calibration curves for DPZ and TAD were plotted with seven concentrations over the range of 1-200 ng/mL. The equation for the calibration curves (n = 5) with the mean ± SD of the slope and the intercept was y = 0.277 (± 0.027) x + 0.033 (±0.007) (r 2 ≥ 0.9904) and y = 0.0402 (± 0.0027) x + 0.001 (±0.003) (r 2 ≥ 0.9966) for DNZ and TAD, respectively. The average slope and intercept of the regression equations were 0.2770 (co-efficient of variation (CV): 8.6%) for DPZ and 0.0403 (CV: 6.0%) for TAD. The linearity was satisfactory and reproducible over time.

Precision and Accuracy
The precision and accuracy of the intra-and inter-day analyses are summarized in Table 1. For DPZ, the intra-day precision ranged from 1.06% to 2.26%, whereas the accuracy ranged from 86.93% to 102.17%. The inter-day precision and accuracy ranged from 3.14% to 4.75% and 89.08% to 102.53%, respectively. TAD, intra-day precision, and accuracy ranged from 1.90% to 5.52% and 95.80% to 99.66%, respectively. The inter-day precision and accuracy ranged from 3.42% to 8.19% and 96.40% to 99.48%, respectively. These

Precision and Accuracy
The precision and accuracy of the intra-and inter-day analyses are summarized in Table 1. For DPZ, the intra-day precision ranged from 1.06% to 2.26%, whereas the accuracy ranged from 86.93% to 102.17%. The inter-day precision and accuracy ranged from 3.14% to 4.75% and 89.08% to 102.53%, respectively. TAD, intra-day precision, and accuracy ranged from 1.90% to 5.52% and 95.80% to 99.66%, respectively. The inter-day precision and accuracy ranged from 3.42% to 8.19% and 96.40% to 99.48%, respectively. These results confirm the accuracy and precision of the simulated quantification method over a wide concentration range of the assay and satisfied the precision and accuracy ranges (%), as specified in the guidance of the MFDS and the FDA for bioanalytical applications [41,42].

Stability
The stability of the stock solutions and rat plasma samples of DPZ and TAD under various conditions is shown in Table 2. The peak area response of the stored samples was directly compared with that of fresh stock solutions. The stability of the stock solution at room temperature (3 h) was 103.43% for DPZ and 107.75% for TAD. In addition, the stock solution stability under refrigeration (4 • C) for 14 days was 103.53% for DPZ and 106.30% for TAD. The plasma stability of DPZ and TAD was assessed by analyzing three replicate samples at two QC levels (2 and 160 ng/mL) under the following storage conditions. The stability of DPZ and TAD in rat plasma was maintained without apparent loss under various storage and handling conditions: room temperature (5 h), refrigeration (4 • C) for 14 days, four freeze/thaw cycles, extraction in an autosampler (10 • C, 20 h), and frozen conditions (−70 • C) for 4 weeks, which implies the long-term stability of DPZ and TAD. Therefore, based on the deviations from the nominal concentration within ±15%, DPZ and TAD were considered stable in rat plasma under all examined conditions without substantial degradation.  Table 3 highlights the recoveries of DPZ and TAD at three QC concentration levels (2, 16, and 160 ng/mL, n = 6), along with IS recovery (20 ng/mL, n = 6). The extraction recovery was evaluated by comparing the mean area responses of the two analytes spiked before extraction with those spiked after extraction. The matrix effect was evaluated by comparing the mean area responses of the two analytes spiked after extraction with neat solution samples at two QC levels (2 and 160 ng/mL, n = 6) for DPZ and TAD. The absolute extraction recoveries for DPZ and TAD were 90.86-95.99% and 96.17-98.59%, respectively. The extraction recovery of IS was 97.96%. The mean extraction recovery for DNA and TAD in rat plasma was greater than 90% at all three tested quality control (QC) concentrations with CVs of 15% or less (Table 3), suggesting high reproducibility and excellent analyte recovery using our one-step sample preparation method with PP.  According to previous studies, the effects of the matrix on extraction recovery, ion enhancement, and ion suppression should be critically assessed to develop a valid bioanalytical method using LC-MS/MS [15,16]. Therefore, the matrix effects for DPZ and TAD were 97.05-108.78% and 56.16-58.46%, respectively. The matrix effect of IS was 90.90%, and no significant differences in peak areas were detected. Even the mean matrix effects for TAD in rat plasma are less than 60%, with the corresponding CV (%) values of less than 15% at the two QC concentrations, suggesting acceptable and reproducible matrix effects between the analyte and endogenous substances in rat plasma.

Analytical Method Implementation to DDI PK Study in Rats
The established LC-MS/MS analytical method was successfully implemented to simultaneously quantify the PK parameters of DPZ and TAD in plasma after oral administration of DPZ and TAD, either alone or in combination in rats. The mean plasma concentrationtime curves of DPZ and TAD are depicted in Figure 3. The PK parameters of DPZ and TAD are summarized in Table 4. The C max of single DPZ administration was 59.776 ng/mL, achieved at a very short T max of 0.5 h with a high AUC last of 129.583 h·ng/mL. C max of DPZ in combination treatment with TAD was 39.235 ng/mL, and the AUC last was 100.5 h·ng/mL, which was also attained in a very short time (T max = 0.5 h). The C max of a single TAD administration was 17.795 ng/mL, and the C max of TAD in combination with DPZ was 13.750 ng/mL. In addition, the AUC last of a single TAD administration was 46.987 h·ng/mL, and the AUC last of the combination was 34.419 h·ng/mL. Both groups exhibited very short T max values (0.5 h). Our test of the longer T 1/2 and MRT last (mean ± SD; 2.630 ± 1.302 h and 2.617 ± 0.923 for single DAZ, respectively, vs. 3.793 ± 2.032 h and 3.735 ± 1.602 h for DPZ in combination with TAD, respectively) were observed. In addition, higher CL/F was found as 7.355 ± 4.516 L/h and 11.819 ± 9.852 L/h for single TAD and for TAD in combination with DPZ, respectively. A comparison of the plasma levels of DPZ and TAD between single and combination administration showed that a single administration of each drug achieved higher values than those of the combination groups. However, the differences in the mean plasma concentration and PK parameters between the single and combination groups of DPZ and TAD were statistically insignificant (p > 0.05). For these reasons, it is difficult to determine the potential DDI between DPZ and TAD at this time. Most PK-related DDIs occur through the induction or inhibition of metabolic enzymes, including CYP 450 and UDP-glucuronosyltransferase or drug transporters [43]. DPZ is primarily metabolized by CYP 2D6 and CYP 3A4, and then undergoes glucuronidation [44,45], whereas TAD is metabolized by CYP 2D9 and CYP 3A4. Although both drugs are metabolized by CYP3A4, PK-related DDI between DPZ and TAD has not been reported. However, more mechanistic studies and multiple dose administration for at least one week for pharmacokinetic or toxicokinetic studies should be conducted to clarify potential DDI secondary to the interference of the metabolic pathways of DPZ and TAD. was 13.750 ng/mL. In addition, the AUClast of a single TAD administration was 46.987 h·ng/mL, and the AUClast of the combination was 34.419 h·ng/mL. Both groups exhibited very short Tmax values (0.5 h). Our test of the longer T1/2 and MRTlast (mean ± SD; 2.630 ± 1.302 h and 2.617 ± 0.923 for single DAZ, respectively, vs. 3.793 ± 2.032 h and 3.735 ± 1.602 h for DPZ in combination with TAD, respectively) were observed. In addition, higher CL/F was found as 7.355 ± 4.516 L/h and 11.819 ± 9.852 L/h for single TAD and for TAD in combination with DPZ, respectively. A comparison of the plasma levels of DPZ and TAD between single and combination administration showed that a single administration of each drug achieved higher values than those of the combination groups. However, the differences in the mean plasma concentration and PK parameters between the single and combination groups of DPZ and TAD were statistically insignificant (p > 0.05). For these reasons, it is difficult to determine the potential DDI between DPZ and TAD at this time. Most PK-related DDIs occur through the induction or inhibition of metabolic enzymes, including CYP 450 and UDP-glucuronosyltransferase or drug transporters [43]. DPZ is primarily metabolized by CYP 2D6 and CYP 3A4, and then undergoes glucuronidation [44,45], whereas TAD is metabolized by CYP 2D9 and CYP 3A4. Although both drugs are metabolized by CYP3A4, PK-related DDI between DPZ and TAD has not been reported. However, more mechanistic studies and multiple dose administration for at least one week for pharmacokinetic or toxicokinetic studies should be conducted to clarify potential DDI secondary to the interference of the metabolic pathways of DPZ and TAD.

Materials
DPZ (purity 99.6%), TAD (purity 99.7%), and lansoprazole (LPZ; internal standard (IS), purity 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water was prepared using an Aqua MAX Ultra 370 (YL Instrument, Gyeonggi-do, Republic of Korea) water purification system for all analyses. For HPLC analysis, methanol was purchased from Burdick & Jackson (Charlotte, NC, USA), and acetonitrile was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ammonium acetate and formic acid were used for the mobile phase buffer preparation. Ammonium acetate and formic acid were obtained from Sigma-Aldrich and from Wako Chemicals (Osaka, Japan), respectively.

Mass Spectrometric Conditions
The optimized MS/MS conditions for simultaneous quantification of DPZ, TAD, and LPZ (IS) were as follows: gas 1:40.0 psig, gas 2:30.0 psig, ion spray voltage: 5500.0 V, turbo heater temperature: 550.0 • C, entrance potential: 10.0 V, collision activation dissociation: 9 psig, curtain gas: 20 psig, cell exit potential: 10 V. The declustering potential and collision energy were applied at 60 and 35 V for DPZ, 100 and 20 V for TAD, and 50 and 20 V for LPZ, respectively. Data on DPZ, TAD, and LPZ were collected and quantified using the Analyst software version 1.6.3. (AB SCIEX, Framingham, MA, USA).

Preparation of Calibration Standards and Plasma Samples
Standard stock solutions of DPZ, TAD, and LPZ (IS) were prepared in methanol at a concentration of 100 µg/mL and stored at −20 • C. Working solutions were prepared by diluting the stock solutions with 50% methanol. The concentration of DPZ and TAD ranged from 1 to 200 ng/mL. Quality control (QC) samples were prepared at four different concentrations (1, 2, 16, and 160 ng/mL). The IS working solution was prepared by diluting the stock solution with 50% methanol to a concentration of 20 ng/mL. All working stock solutions were prepared and stored at −20 • C until use. Calibration standards and QC samples of DPZ and TAD in rat plasma were diluted with the corresponding working solutions and blank rat plasma. Drug-free plasma, that is, control (blank) plasma, stored in a deep freezer, was completely thawed at room temperature before use. Ten percent spiking with working DPZ and TAD stock solutions was performed in blank plasma. The spiked QC samples were stored at −70 • C for stability studies.

Preparation of Plasma Samples
The rat plasma was stored at −70 • C. The plasma samples were thawed at room temperature and then centrifuged at 1800× g (4 • C) for 8 min before pipetting. Using a micropipette, 30 µL of the plasma sample was transferred into a polypropylene microtube, and 10 µL of IS working solution was added to each sample. Further, 0.2 mL of acetonitrile was then added and vortexed for 1 min. The tube was then centrifuged at 22,285× g (4 • C) for 5 min. The supernatant was transferred to an analytical vessel, and 2 µL of the sample was injected into the LC-MS/MS system.

Protocol Validation
The established methods were validated by the required bioanalytical method validation guidelines published by the Ministry of Food and Drug Safety in Korea (MFDS) and the U.S. Food and Drug Administration (FDA) [26,27]. The established protocol was validated for selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability in compliance with the bioanalytical method guidelines.
3.6.1. Specificity and Lower Limit of Quantitation (LLOQ) Selectivity was examined by analyzing six randomly selected blank rat plasma samples to investigate the presence of endogenous materials. These samples were prepared using the pretreatment procedure described in Section 3.5, and both the analytes and IS were free of interference with each other. LLOQ implies the lowest concentration of the analyte in rat plasma. The signal-to-noise (S/N) ratio was greater than 10, and the deviation from accuracy and precision was less than 20%.

Linearity
Linearity was determined using the linear regression model (1/x 2 ) for the calibration curve range of DPZ and TAD. The calibration curves were expressed as y = ax + b, where y represents the mean of the peak area ratios of the analytes to their internal standards, a represents the slope of the calibration curves, b represents the y-intercept of the calibration curve, and x represents the analyte concentration. Satisfactory linearity was determined using a correlation coefficient of 0.99 or greater.

Precision and Accuracy
The precision and accuracy of the simulated quantification method were determined by concentration analysis of five replicates of each sample from the validation batch. For intra-and inter-day testing, four different concentrations of QC samples were prepared, and five sets of repeated sets were analyzed to determine the accuracy and precision for three consecutive days. The accuracy and precision of within-and between-run analyses should be within 15% of the nominal values, except for the LLOQ, which should be within 20% of the nominal value.

Stability
Stability analyses of the stock solutions were performed at room temperature for 3 h and under refrigerated conditions (4 • C) for 14 days. The stability of the plasma analytes was tested for low and high QC under pre-specified conditions: room temperature for 5 h, refrigeration (4 • C) for 14 days, autosampler (10 • C) for 20 h, four cycles of freezing-thawing, and finally frozen conditions (−70 • C) for 28 days for long-term stability.

Recovery and Matrix Effect
Recovery and matrix effects were measured by evaluating matrix-induced ion enhancement or suppression. The recovery was examined at three QC concentrations by comparing the mean analytical peak area of six replicates of each QC sample and the corresponding areas of the equivalent concentration of the analytes spiked into blank rat plasma after extraction. The matrix effect was determined by comparing the peak area of standard QC concentrations with the corresponding areas of the equivalent concentrations of the spiked analytes extracted from the blank rat plasma.

Application to a Pharmacokinetic Study
The pharmacokinetic (PK) tests in rats were approved by the Animal Ethics Committee (PK Protocol number: 21-KE-155; date of approval: March 2, 2021). Male Sprague-Dawley rats (5 weeks old) from KNOTUS Co., Ltd. (Incheon, Republic of Korea) were maintained under consistent laboratory conditions (temperature, 23 ± 3 • C; relative humidity, 55 ± 15%; light/dark cycle, 12 h). Rats were divided into three groups (n = 3): group 1, DPZ (3 mg/kg); group 2, TAD (0.3 mg/kg); and group 3, DPZ (3 mg/kg) combined with TAD (0.3 mg/kg). Animals were fasted overnight and allowed free access to water. DPZ powder and TAD powder in aqueous solutions were orally administered to rats at an equivalent DPZ dose of 2.26 mg/kg and TAD dose of 2.25 mg/kg (3 and 0.3 mg daily dosage converted to volume ratio, respectively. Blood samples (300 µL) were directly collected from the jugular vein using heparinized syringes at 0.5, 1, 2, 4, 6, 8, and 24 h. The collected blood samples were immediately centrifuged at 19,314× g for 2 min to prepare plasma samples, which were then stored at −70 • C until analysis.

Statistical Analysis of the Pharmacokinetic Study
The peak plasma concentration, C max , and T max (the time to reach C max ) for DPZ and TAD were calculated using individual plasma concentration-time profiles. A noncompartmental method for extravascular input provided in BA Cal 2007 software (MFDS, ver. 1.0.0) was used to calculate PK parameters, including AUC last (area under the plasma drug concentration-time curve between 0 and 24 h) and extrapolated AUC inf (AUC from 0 to infinity). The terminal half-life (T 1/2 ) was calculated as ln 2/λ z (λz is the elimination rate constant estimated by using the least-squares linear regression method of the terminal log-linear portion of the plasma concentration-time curve). The apparent clearance (CL/F) was calculated as CL/F = dose/AUC 0-∞. The Mann-Whitney U test was used to determine the differences in the mean plasma concentration and PK profiles of DPZ and TAD between the single and combined administration groups. Statistical analysis was performed using SPSS Statistics (version 26.0; IBM SPSS Statistics for Windows, Armonk, NY, USA), and a p-value < 0.05 was considered statistically significant.

Conclusions
We established the first sensitive and simple LC-MS/MS-based analytical method for simultaneously quantifying DPZ and TAD in rat plasma. This LC-MS/MS analytical method was fully validated according to the guidelines published by the MFDS in Korea and the U.S. FDA and was successfully implemented in a PK study designed for DDI detection between DPZ and TAD in anticipation of their potential concurrent clinical use for AD management. Our analytical method for simultaneous quantification of DPZ and TAD achieved excellent performance and offered the following advantages over other previously reported single analyte quantification methods of either DPZ or TAD: the analysis was performed using a small plasma volume (30 µL) and a simple and rapid protein precipitation protocol, that is, high-throughput [27,[35][36][37][46][47][48][49][50]. This study provided the first preliminary evidence of non-significant DDI between DPZ and TAD based on PK profiles in rats. The findings of this study may contribute to the current body of literature, especially for therapeutic drug monitoring associated with DDI; however, further detailed studies on the PK parameters of DPZ and TAD are still warranted to determine risks versus benefits, if any, associated with their concurrent administration.

Data Availability Statement:
The data presented in this study are available upon request.