Optimized method development and validation for determining donepezil in rat plasma: A liquid-liquid extraction, LC-MS/MS, and design of experiments approach – PubMed Black Hawk Supplements

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Donepezil (DPZ), a piperidine-based reversible cholinesterase inhibitor, finds extensive use in treating Alzheimer’s disease (AD). Originally designed as an oral formulation, DPZ encounters drawbacks such as a brief duration of action and reduced treatment effectiveness in elderly patients with memory impairment or difficulty swallowing medications. To address these issues and improve patient compliance, researchers are actively exploring alternative DPZ formulations. Consequently, reliable…
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Optimized method development and validation for determining donepezil in rat plasma: A liquid-liquid extraction, LC-MS/MS, and design of experiments approach - PubMed

Optimized method development and validation for determining donepezil in rat plasma: A liquid-liquid extraction, LC-MS/MS, and design of experiments approach

Ye Bin Shin et al. PLoS One. .

Abstract

Donepezil (DPZ), a piperidine-based reversible cholinesterase inhibitor, finds extensive use in treating Alzheimer’s disease (AD). Originally designed as an oral formulation, DPZ encounters drawbacks such as a brief duration of action and reduced treatment effectiveness in elderly patients with memory impairment or difficulty swallowing medications. To address these issues and improve patient compliance, researchers are actively exploring alternative DPZ formulations. Consequently, reliable methods are necessary to quantitate DPZ in biological samples for in vivo assessment. Therefore, we propose an efficient, sensitive, wide-dynamic, and cost-effective method for quantitating DPZ in rat plasma. Our method employs liquid-liquid extraction (LLE) followed by liquid chromatography and tandem mass spectrometry, enabling in vivo evaluation of novel DPZ formulations. Notably, our method requires only 20 μL of rat plasma and employs icopezil as the internal standard-a cost-effective compound with chemical similarity to DPZ. We meticulously optimized LLE conditions, taking into account factor interactions through design of experiments (DOE). Our rapid and straightforward extraction and purification involved using 500 μL of pure methyl tert-butyl ether to extract DPZ from the sample within five minutes. The dynamic range of the method extends from 0.5 ng/mL to 1,000 ng/mL, demonstrating excellent sensitivity and suitability for pharmacokinetic studies across diverse DPZ formulations. Following the FDA guidelines, we rigorously validated the developed method, evaluating selectivity, linearity (with a coefficient of determination ≥0.9999), accuracy (ranging from 96.0% to 109.6%), precision (≤13.9%), matrix effect (92.2% to 103.8%), recovery (98.5% to 106.8%), the lower limit of quantitation (0.5 ng/mL), and stability. Finally, we effectively employed the validated method for the long-term pharmacokinetic assessment of a DPZ formulation. We expect that this approach will make a substantial contribution to the advancement of new DPZ formulations, ultimately benefiting individuals afflicted by AD.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1

Chemical structures of donepezil (A) and icopezil (B).

Fig 2
Fig 2

Multiple reaction monitoring chromatograms of donepezil (DPZ, left panels) and icopezil (internal standard, IS, right panels) in rat plasma: Double blank plasma (A), blank plasma with 100 ng/mL of the IS (B), and plasma with 0.5 ng/mL of DPZ and 100 ng/mL of the IS (C).

Fig 3
Fig 3

Pareto chart of the effects for extraction efficiencies of donepezil: Volumetric ratio of methyl tert-butyl ether to ethyl acetate in the extraction solvent (%, A), volume of the extraction solvent (μL, B), extraction duration (minutes, C), and extraction temperature (°C, D).

Fig 4
Fig 4

Response surface plots for effects of volumetric ratio of methyl tert-butyl ether to ethyl acetate in the extraction solvent (%, A), volume of the extraction solvent (μL, B), and extraction duration (minutes, C) on extraction efficiency.

Fig 5
Fig 5. Plasma concentration-time profile of donepezil (DPZ) following intramuscular injection of drug suspension in normal rats (24.5 mg/kg as DPZ).

Data represent mean ± standard deviation (n = 5).

References

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Optimized method development and validation for determining donepezil in rat plasma: A liquid-liquid extraction, LC-MS/MS, and design of experiments approach – PubMed