Abstract
Aim: A HPLC method was developed and validated for the novel combination of rutin (RN) and donepezil (DNP). Materials & methods: RN and DNP were simultaneously eluted through a C18 column (Ø 150 × 4.6 mm) with a 60:40 v/v ratio of 0.1% formic acid aqueous solution to methanol at 0.5 ml/min. Results: The purposed method was found linear, selective, reproducible, accurate and precise with percent RSD less than 2. The limit of quantification for RN and DNP was found 3.66 and 3.25 μg/ml, respectively. Conclusion: Validated as per the ICH guidelines, the developed method efficiently quantified RN and DNP co-loaded in DQAsomes (121 nm) estimating matrix effect, release profile, entrapment efficiency, loading efficiency and in vivo plasma kinetics.
Keywords: : donepezil, DQAsomes, HPLC, PKSolver, rutin, simultaneous estimation
GRAPHICAL ABSTRACT
Plain language summary
Summary points.
A simple, robust and economical RP-HPLC method was developed to simultaneously estimate rutin (RN) and donepezil (DNP) and validated as per International Conference of Harmonization (ICH) regulatory guideline.
The limit of detection (LOD) was found to be 1.209 μg/ml and 1.074 μg/ml, while the Limit of Quantification (LOQ) was found to be 3.664 μg/ml and 3.257 μg/ml for RN and DNP, respectively.
Artificial cerebrospinal fluid (ACSF) matrix showed ion suppression for both RN and DNP whereas dequalinium (DQA) as formulation matrix demonstrated ion suppression for RN and ion enhancement for DNP.
RN and DNP co-loaded DQAsomes was prepared and estimated for loading efficiency, entrapment efficiency, release profile and plasma kinetics providing practical implication of developed method in both pharmaceuticals and biological samples.
1. Background
With increase in aging population round the globe, Alzheimer's disease (AD) being the commonest form of dementia has promptly raised the public health concern [1]. Long before the symptoms of AD show up, a series of pathophysiological changes occur and proceed in a predictable manner [2]. One such changes, following the amyloid cascade hypothesis, is extracellular Aβ deposition, which may aggravate in parallel a cascade of events including cognitive decline. Consequently, a number of anti-amyloid monotherapies have been developed but failed to translate due to their inefficiency to reduce the progression of cognitive decline. Furthermore, recently developed anti-amyloid therapies such as aducanumab, when administered in early stages of AD have limited cognitive decline to an extent, but could have been more effective when combined with an agent specific to cognition. Thus, combination therapies have gained interest [2,3], and regulation across countries has been set by International Conference of Harmonization (ICH). In view of the aforementioned consequences, rutin (quercetin-3-rutinoside) (RN) a natural flavonoid with known amyloid clearance activity [4–7] when combined with an agent that reduces cognitive decline such as donepezil (DNP) might embark state of the art in therapy against progression of neurodegenerative diseases such as AD. Since RN is hydrophobic and DNP is hydrophilic in nature, a number of analytical and bioanalytical methods for estimation of either RN or DNP through high-performance liquid chromatography (HPLC) have already been developed, but the combination is not yet explored. Ishii et al. developed a bioanalytical method for RN by HPLC using acetonitrile and 10 mM ammonium acetate solution with 0.3 mM EDTA-glacial acetic acid in the ratio of 16.5:82.5 v/v (pH 3.8) as mobile phase [8]. Zu et al. [9] developed method for simultaneous estimation of catechin, RN, quercetin, kaempferol and isorhamnetin in the extract of sea buckthorn leaves against 1.0% acetic acid solution containing methanol:acetonitrile:water in the ratio of 40:15:45, v/v/v as mobile phase. Further, Pappa et al. [10] developed and validated an analytical method for the estimation of DNP in tablets through HPLC using reversed phase (RP) C18 column with a 50:50:0.5 v/v/v ratio of methanol, phosphate buffer basified with triethylamine as mobile phase. DNP was eluted at 9 min. Ragab and Bahgat [11] simultaneously estimated DNP and citalopram in both tablet and plasma matrix using a ratio 65:35 v/v of phosphate buffer (0.05 M) and acetonitrile on C18 column. DNP was eluted at 4.5 min. Thus, considering contrasting solubility of RN and DNP as a challenge to simultaneously estimate, the purposed research work aims to develop a robust and economical method for simultaneous determination of RN and DNP as per ICH guidelines [12,13] and their application in the matrix of nanocarrier and plasma. The developed method would generate a proof of concept and relevance for simultaneous estimation of RN and DNP in lipid-based delivery system, DQAsomes [14]. To prove the application of the developed method, RN and DNP have been co-loaded inside lipid-based formulation and characterized for drug release, loading, entrapment efficiency and pharmacokinetic study on C57BL/6 adult male mice [15,16].
2. Experimental
2.1. Equipment
The binary modular chromatographic system SIL-20AC HT (Shimadzu corporation, Japan) included Lab solution software (Shimadzu Corporation, Japan), binary LC20DA pump (Shimadzu Corporation, Japan), rheodyne injector model 7125, 20 μl loop, column oven CT0-10AS VP, SPD-M20A UV-PDA detector (Shimadzu Corporation, Japan), and C18 column (Ø 150 × 4.6 mm, 4 μm, Thermo Fisher Scientific, USA).
2.2. Reagents & materials
RN was procured from TCI Chemicals India Pvt. Ltd and DNP was obtained as a gift sample from Lupin Ltd, Pune, India. Methanol (MH), acetonitrile (ACN), ethanol (EH), ethyl acetate (EA), chloroform (CF), n-hexane (HEX), tert butyl methyl ether (TBME), diethyl ether (DEE) and formic acid (FA) were of HPLC grade, and purchased from Merck, Mumbai, India. Triple distilled water (TDW) was prepared by a Milli-Q water purification system (Millipore, MA, USA). Dequalinium (DQA) was purchased from Sigma Aldrich, USA.
2.3. Chromatographic conditions
Chromatographic analysis of RN and DNP was carried out using C18 reversed-phase column (Ø 150 × 4.6 mm, Thermo Fisher Scientific, USA), packed with 4 μm diameter particles. 0.1% FA aqueous solution, adjusted to pH = 3 and MH, filtered through 0.45 μm membrane filter (Millipore), and deaerated ultrasonically was used as mobile phase in the ratio 60:40 v/v at 0.5 ml/min. Column oven temperature was maintained at 40°C. RN and DNP were quantified by PDA detector following separation through RP-HPLC at 355 and 268 nm, respectively. The total analysis run time was set to 10 min. ACN, MH and TDW in the ratio 40:40:20 v/v was used as rinsing solvent.
2.4. Preparation of stock solutions for quality control samples
An accurately weighed quantity of RN and DNP was dissolved in MH to prepare a stock solution of the combination (RD) at a concentration of 1 mg/ml. This stock solution was further diluted to a concentration of 100 μg/ml using TDW to prepare a secondary stock solution. The prepared stock solutions were stored at -80°C until further use. Additionally, the required working dilutions for quality control (QC) were prepared from the stock solution immediately before analysis using TDW as the diluent.
2.5. Preparation of calibration standard & quality control samples
Working solution of RD was prepared from stock solution by diluting it in TDW, at a concentration ranging from 0.195 μg/ml to 100 μg/ml for the calibration plot (CP) and 5 μg/ml, 10 μg/ml and 15 μg/ml representing QC samples at low, middle and high levels, respectively. All the samples were freshly prepared and filtered through a 0.45 μm membrane filter (Millipore) and injected using autosampler.
2.6. Preparation of calibration standard & quality control plasma samples
For the estimation of RN and DNP in plasma, CP with a final concentration ranging from 0.195 μg/ml to 100 μg/ml were prepared by spiking 10 μl appropriate working solution to 90 μl blank mouse plasma. Furthermore, QC samples in plasma were prepared by spiking an appropriate working solution to plasma to achieve final concentrations of 5 μg/ml, 10 μg/ml and 15 μg/ml representing low, middle and high level respectively.
2.7. Processing of plasma samples
RN and DNP were extracted from plasma using a one-step precipitation method. 10 μl of appropriate sample (RD) was spiked in 90 μl of blank mouse plasma and vortexed. Subsequently, 1 ml of extracting solvent (MH) was added to the spiked plasma samples and vortex-mixed for 10 min to extract RN and DNP and centrifuged at 7000 × g for 5 min at 12°C (Eppendorf, Hamburg, Germany). The clear supernatant was dried using nitrogen evaporator at 40°C, 20 psi and reconstituted in 100 μl TDW. Further, the samples were injected in HPLC using autosampler for analysis. All samples during analysis were maintained at refrigerated condition (2–8°C).
2.8. Method validation
The developed method was validated following international council for harmonization of technical requirements for pharmaceuticals for human use (ICH) guidelines (Q2A, Q2B and M10) [12,13] for analytical and plasma samples respectively. Validation was performed for parameters including system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), selectivity, accuracy, precision, robustness and stability studies under varied storage conditions.
2.8.1. System suitability
The system suitability (SST) for the simultaneous estimation of RN and DNP on the purpose-built HPLC system using the developed method was identified through multiple injections of standard solution of quality control RD samples (n = 6). Stable RT, resolution, tailing factor and theoretical plate were considered as evaluating parameters.
2.8.2. Specificity
The specificity of the developed method was determined in DQA as a formulation excipient, plasma and artificial cerebrospinal fluid (ACSF). Consequently, DQA, plasma and ACSF spiked with and without RD, equivalent to 10 μg/ml was prepared. Further, the specificity was studied in terms of any interference of formulation excipients, plasma and ACSF matrix with peaks of RN and DNP at respective absorption maxima.
2.8.3. Linearity, LOD & LOQ
CP for both analytical and plasma samples were injected into the HPLC. The calibration curve was plotted with peak areas against CP concentration and a weighted (1/x2) linear regression analysis for linearity. It demonstrates a working relation between peak areas obtained for the concentration of RN and DNP injected into HPLC.
The minimum concentration of RN and DNP with reference to CP that can be detected in a sample with statistical accuracy are referred as LOD whereas the lowest concentration of RN and DNP that can be quantified with justified degree of accuracy and precision are called LOQ and calculated following equation.
2.8.4. Accuracy & precision
For analyzing the accuracy of the developed method, QC at low (50%), middle (100%) and high (150%) level were prepared for analytical and plasma samples.
The precision of the developed method was evaluated to study the reproducibility of peak areas of QC sample at a concentration of 10 μg/ml, injected at intermediate level (inter- and intra-day) for 3 consecutive days. The samples (n = 3) were analyzed for percent recovery (%Re) and percent relative standard deviation (%RSD).
2.8.5. Robustness
The robustness of the developed method was estimated by deliberately varying the chromatographic conditions. QC standard and plasma spiked samples (n = 3) were analyzed for changes in flow rate, ratio of mobile phase, and column temperature at a variation of ±10%. The %Re was estimated and the effect on peak area and shift in RT was noted.
2.8.6. Matrix effect
Cerebrospinal spinal fluid (CSF) and plasma are often used to estimate the pharmacokinetic and pharmacodynamic profiles of a central nervous system (CNS) active drugs [17]. Herein, withdrawing CSF in large volume without contamination is challenging thus, artificial CSF (ACSF) is used to study the matrix effect [18]. Possible interference of endogenous substance in plasma, and ACSF with RD were evaluated quantitively using post-extraction method. The matrix effect was calculated by comparing CP of RN and DNP in standard solution with the samples spiked in ACSF, and plasma by following equation.
Lower slope suggests ion suppression whereas higher slope for matrix matched slope indicates ion enhancement [19,20].
2.8.7. Solution stability
The stability of the RN and DNP in standard solution and when spiked in plasma was assessed under varied condition. The QC samples in standard solution were analyzed at 4°C and 25°C and the freeze–thaw stability of plasma spiked QC samples was assessed at 4°C and -20°C. The samples stored under varied temperatures were analyzed for 12, 24 and 48 h. The ratio of stored QC samples was compared with freshly prepared QC samples and studied for percent change in concentration.
2.9. Application of the developed method
2.9.1. Development of RN & DNP co-loaded vesicular nanoformulation
Vesicular nanoformulation (DQAsomes) was synthesized by the conventional thin film hydration method using cationic dequalinium chloride (DQA) [21–23]. Concisely, 10 mM of DQA and RN (5 mg/ml) were dissolved in MH and evaporated at 45°C using rota-evaporator to form a thin film. The film was dried under vacuum for 24 h and hydrated with DNP (5 mg/ml) in 5 mM of HEPES buffer and probe sonicated until a clear solution was obtained.
RN and DNP were extracted from RN and DNP co-loaded vesicular nanoformulation using MH and analyzed for specificity of the developed analytical method in the presence of formulation excipient.
For drug loading and entrapment assay, the developed vesicular nanoformulation was centrifuged at 10,000 g for 10 min [24]. The Pellets were washed with water and disrupted with MH to release the loaded RN and DNP into the solution. The solution was again centrifuged at 10,000 × g for 10 min and the supernatant was analyzed using developed RP-HPLC method. The obtained area under curve (AUC) was processed as percent fraction of total amount of excipients used in DQAsomes for loading efficiency and total theoretical amount of RN and DNP co-loaded in DQAsomes for encapsulation efficiency.
Furthermore, RN and DNP co-loaded DQAsome was studied for their release profile in PBS (pH = 7.4), mimicking physiological conditions using the dialysis method [25–27]. 1 ml of the sample was filled in the dialysis bag and soused inside PBS with 0.1% Tween-80 (to dissolve RN), maintained at 37°C. Aliquots of 1 ml from the dissolution media were collected at predetermined intervals and replenished with an equal volume of fresh PBS to maintain the sink condition. The samples were processed and analyzed using the developed method through HPLC.
2.9.2. In vivo plasma kinetic study
The significance of the developed HPLC method was additionally evaluated through a pharmacokinetics study of RN and DNP co-loaded formulation on administration to adult C57BL/6 male mice (20–22 g, 5–6 weeks) following the Institutional Animal Ethics Committee (IAEC) approval (IAEC/2022/101/Renew-0/Sr.no.4). The animals were habituated at 23–25°C and 50–60% RH under artificial light with a photoperiod maintained at a 12 h light/dark cycle, and provided with water and food ad libitum. The animals were divided into three groups (n = 4 each) and intravenously administered saline, free RD and RN and DNP co-loaded DQAsomes (DRD). RD and DRD were administered intravenously, after overnight fasting at a dose level equivalent to 10 mg/kg and 4 mg/kg for RN and DNP, respectively. Blood samples (0.1–0.2 ml) were collected from the retro orbital plexus of mice at predetermined interval [28]. The plasma was further isolated by spinning blood samples at 3000 rpm for 10 min, maintaining 4°C. The obtained plasma samples were stored at -80°C until analysis through HPLC. The plasma samples on the day of analysis were processed following the procedure mentioned in section 2.7. By computing the peak area, various pharmacokinetic parameters were calculated by fitting a non-compartmental model (i.v. bolus) in the Microsoft Excel add-in program, PK Solver [29].
3. Results & discussion
3.1. Optimization
3.1.1. Method development
Contrary to the hydrophilicity of DNP, RN has the least aqueous solubility. Thus, the method was critically optimized for their separation at their respective maximum wavelength (λmax) with requisite resolution and analysis by changing the chromatographic parameters. Both analytes yielded better resolution on C18 column (Ø 150 × 4.6 mm, 4μm, Thermo Fisher scientific, USA), maintained at 40°C against 0.1% v/v aqueous solution of FA maintained at pH 3 and MH in the ratio of 60:40 at 0.5 ml/min. Furthermore, the developed method exhibited simultaneous estimation of RN and DNP in a reproducible manner at retention times (RT) 5.45 min and 7.5 min, respectively (Figure 1).
Figure 1.
(A) Peak identification and purity of (B) rutin and (C) donepezil in standard solution.
3.1.2. Plasma sample extraction procedure
RN and DNP bounded with plasma proteins were extracted through conventional protein precipitation method. Various solvents including MH, ACN, EA, HEX, TBME and DEE were screened as extracting solvent. However, MH yielded clean extracts of RN and DNP with optimum recovery and no significant interference of matrix at their respective retention times was observed. The peaks of RN and DNP were identified at RT 5.5 and 7.2 min, respectively, demonstrating a slight shift indicating the effect of the plasma matrix.
3.2. Method validation
The developed method for the simultaneous estimation of RN and DNP was validated for system suitability, linearity, LOD, LOQ, accuracy, precision, robustness and stability. The percent RSD of the validated parameters was calculated and found to be within the acceptable range (<5% for bioanalytical samples).
3.2.1. System suitability
Before validating the developed analytical method, the system suitability needs to be checked to avoid any disturbances. SST for RN and DNP was found acceptable, with a resolution greater than 6, tailing factor less than 2, and theoretical plate greater than 5000. Both analytes were found stable at RT 5.45 ± 0.2 min (for RN) and 7.5 ± 0.2 min (for DNP) on multiple injection (Figure 3C).
Figure 3.
(A) Calibration plot (B) overlay chromatogram and (C) system suitability of rutin and donepezil in standard solution.
DNP: Donepezil; RN: Rutin.
3.2.2. Specificity
Standard solutions of RN and DNP were prepared in methanol (1 mg/ml) and spiked into plasma, ACSF and DQA at a concentration of 10 μg/ml to assess the specificity of the developed method. As depicted in Figure 2, no interference with the respective peaks of RN and DNP was found. However, the retention time was slightly changed for plasma spiked samples in comparison to the standard solution at absorption maxima 268 and 355 nm, respectively.
Figure 2.
Specificity of developed method for simultaneous estimation of rutin and donepezil in (A) plasma (B) ACSF and (C) DQA matrices.
ACSF: Artificial cerebrospinal spinal fluid; DNP: Donepezil; RN: Rutin.
3.2.3. Linearity, LOD & LOQ
A series of standard solutions and plasma-spiked samples of RN and DNP were analyzed to predict the linearity between their standard concentrations and average peak areas by plotting a graph. The linear regression equations for RN and DNP in standard solution were found to be y = 13212x-2647, r2 = 0.9999 and y = 9927x-3445, r2 = 0.9998, respectively (Figure 3A & B), where ‘x’ represents the concentration, and ‘y’ represents the peak area of standard solution of RN and DNP, respectively. Likewise, in plasma, the regression equation was found to be y = 2540x + 424.1, r2 = 0.9966 and y = 2105x + 2227, r2 = 0.9919, respectively (Supplementary Figure S1).
Further, the LOD and LOQ values for both the drugs were computed mathematically in accordance with ICH guidelines, using residual standard deviation (σ) and slope (S) of the calibration curve plotted, following the equation mentioned in section 2.8.3. Different regression analysis parameters for RN and DNP standard solutions are mentioned in Table 1. The LOQ values of RN and DNP was found 1.404 and 1.573 μg/ml in DQA, 6.795 and 6.629 μg/ml in plasma and 7.446 and 5.717 μg/ml in ACSF matrices, respectively.
Table 1.
Linear regression parameter of rutin and donepezil.
| Parameters | Rutin | Donepezil |
|---|---|---|
| Range of calibration curve (μg/ml) | 0.195–100 | 0.195–100 |
| Detection wavelength (nm) | 355 | 268 |
| Retention time (minute) | 5.45 | 7.5 |
| Correlation coefficient (r2) | 0.9998 | 0.9999 |
| Slope | 9927 | 13212 |
| Intercept | -3445 | -2647 |
| LOQ (μg/ml) | 3.664 | 3.257 |
| LOD (μg/ml) | 1.209 | 1.074 |
3.2.4. Accuracy & precision
To assess the accuracy and precision of the developed method, the QC samples at lower, middle and higher level of both the standard solution and plasma spiked RN and DNP were analyzed for %Re and %RSD. The variations were found less than 2% for standard samples and 5% for plasma spiked samples. The %Re to estimate accuracy was found in the range of 100–102% for RN and 98–102% for DNP justifies the accuracy of the develop method (Supplementary Tables S1 & S2). Further, the %RSD of RN and DNP at both inter- and intra-day (Supplementary Tables S3 & S4) demonstrates precision of the developed method. Peaks at 2.5 min and 3.2 min in plasma spiked samples represented their related matrix.
3.2.5. Robustness
The deliberate change in chromatographic parameters for the developed method defines its reliability even under varied conditions. The percent recovery of RN and DNP with changed parameter has been tabulated in Supplementary Table S5. During the course of analysis, the retention time was noted to be considerably altered while changing the composition of mobile phase and flow rate, and a minor deflection was observed while changing the column temperature. However, the peak area remained consistent for the tested concentration under varied condition. Percent recovery for RN was found in the range of 97.87–102.92% and for DNP it was within 98.27–102.12% with percent RSD less than 5 under varied chromatographic conditions.
3.2.6. Matrix effect
No intrusive peaks were found interfering at the retention time for the formulation and plasma as compared with the standard solution. The recovery of RN and DNP in plasma samples was found to be 70% using the precipitation method. Additionally, the matrix effect of the DQA used to formulate DQAsomes and ACSF in the estimation of RN and DNP was quantitated by plotting CP (Supplementary Figure S2). The deviation in the slope of spiked sample was compared with the standard solution and was found to be within the acceptable range (85–115%). Furthermore, ACSF showed 92.82% and 87.97% ME whereas DQA displayed 91.76% and 103.41% ME for RN and DNP respectively. Thus, ACSF showed ion suppression for both RN and DNP whereas DQA demonstrated ion suppression for RN and ion enhancement for DNP [30,31].
3.2.7. Short-term solution stability
The stability study was performed at 4°C and 25°C for standard QC samples whereas the stability of plasma spiked QC samples was analyzed at 4°C and -20°C for 48 h. The processed samples for stability were compared with the freshly processed samples. QC samples in the standard solution were found to be stable at both refrigerated and room temperature even after 48 h. Furthermore, the plasma spiked QC samples undergoing freeze-thaw stability were also found to be stable up to 48 h (Table 2).
Table 2.
Solution stability of standard solution of donepezil and rutin at 4°C and 25°C, measured in terms of %Recovery and %RSD.
| Temp. (°C) | Time (h) | Conc. (μg/ml) | Donepezil | Rutin | ||
|---|---|---|---|---|---|---|
| %Re | %RSD | %Re | %RSD | |||
| Standard QC sample | ||||||
| 4 | 12 | 10 | 102.89 | 0.53 | 98.38 | 2.56 |
| 24 | 10 | 101.58 | 1.82 | 99.2 | 1.73 | |
| 48 | 10 | 101.29 | 0.14 | 98.71 | 0.73 | |
| 25 | 12 | 10 | 102.60 | 0.02 | 98.39 | 1.93 |
| 24 | 10 | 99.92 | 0.02 | 99.66 | 0.27 | |
| 48 | 10 | 99.88 | 0.10 | 98.46 | 0.93 | |
| Plasma QC sample | ||||||
| 4 | 12 | 10 | 100.08 | 1.06 | 101.47 | 0.76 |
| 24 | 10 | 99.67 | 0.96 | 101.43 | 1.27 | |
| 48 | 10 | 100.36 | 2.97 | 98.22 | 1.16 | |
| -20 | 12 | 10 | 101.2 | 0.28 | 100.22 | 5.4 |
| 24 | 10 | 100.52 | 0.09 | 101.86 | 1.49 | |
| 48 | 10 | 96.97 | 1.54 | 99.41 | 1.00 | |
3.3. Application of the developed method
3.3.1. Characterization of developed vesicular nanoformulation
DQAsomes of 121 nm with PDI 0.34 and surface zeta potential -11.8 mV was developed. To demonstrate the applicability of the developed method, RN and DNP loaded DQAsomes were prepared and characterized for specificity, loading efficiency and entrapment efficiency. No significant interference from the formulation matrix was observed when co-loaded with drugs as depicted in the chromatogram (Supplementary Figure S3). The specificity, drug release, loading efficiency and entrapment efficiency of the developed DQAsomes (Figure 4) validate the selectivity and utility of the developed method for the simultaneous estimation of RN and DNP.
Figure 4.
(A) Size (B) zeta potential and (C) percent loading efficiency, (D) percent entrapment efficiency and (E) drug-release profile of developed RN and DNP co-loaded DQAsomes.
****Indicates the extremely significant difference with p-value < 0.0001.
DNP: Donepezil; RN: Rutin.
3.3.2. In vivo plasma-kinetic study
The developed method was further utilized to study the plasma-kinetic profile of the vesicular nanoformulation when intravenously administered in adult C57BL/6 mice. The resultant concentration-time profiles were estimated using the linear up/ log down method, fitting a non-compartmental model (i.v. bolus) in PKSolver 2.0 (Figure 5). Additionally, the pharmacokinetic parameters of DRD was compared with free RD, tabulated in Table 3. The half-life of RN was found to be increased by 3.23-fold in DRD_RN compared with free RD_RN whereas it increased by 1.62-fold in DRD_DNP as compared with free RD_DNP. This led to an increase in bioavailability by 5.04-fold for RN and 1.43-fold for DNP when encapsulated inside DQA against free RN and DNP solution. Furthermore, a substantial decrease in clearance was observed in RN and DNP co-loaded DQAsomes.
Figure 5.
In vivo plasma kinetic of rutin and DNP co-loaded in DQAsomes (DRD) and free RN and DNP (RD) intravenously administered at a dose of 10 mg/kg and 4 mg/kg respectively to C57BL/6 mice.
DNP: Donepezil; RN: Rutin.
Table 3.
Plasma kinetic profile of rutin and donepezil co-loaded in DQAsomes (DRD) compared with free form of RN and DNP (RD) at 10 mg/kg and 4 mg/kg respectively providing application of the developed method.
| Pharmacokinetic parameter | DRD_RN | DRD_DNP | RD_RN | RD_DNP |
|---|---|---|---|---|
| Cmax (μg/ml) | 40.80 | 20.97 | 30.69 | 11.53 |
| t1/2 (h) | 76.28 | 110.67 | 21.13 | 67.82 |
| Clearance (ml/h/kg) | 0.008 | 0.004 | 0.048 | 0.006 |
| AUC0-∞ (h μg/ml) | 1133.10 | 922.94 | 207.42 | 647.93 |
| Mean residence time (h) | 103.55 | 152.82 | 40.39 | 92.31 |
DNP: Donepezil; RN: Rutin.
4. Conclusion
Hence, a simple and economical HPLC-UV method has been developed for the simultaneous estimation of RN and DNP and validated following ICH guidelines. The purposed method was found linear, selective, reproducible, robust and precise for co-eluting RN and DNP within 10 min at better resolution in both standard form as well as in plasma with percent RSD less than 2 and 5, respectively. Furthermore, the validated method was used to quantify RN and DNP when co-loaded inside the vesicular formulation for their release profile, entrapment efficiency, loading efficiency and in vivo plasma kinetic. No interference in the peaks of respective elutes was found. Thus, attesting to the practical use of the developed method in analytical profiling of RN and DNP when used as a combination therapy for neurodegenerative disease, formulated in different dosage form.
Supplementary Material
Supplemental material
Supplemental data for this article can be accessed at https://doi.org/10.1080/17576180.2024.2344395
Financial disclosure
The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Competing interests disclosure
The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.
Writing disclosure
No writing assistance was utilized in the production of this manuscript. This is CSIR-CDRI communication 10776.
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