Simultaneous HPLC-UV quantification of donepezil and rivastigmine in human plasma using an SBA-15/graphene oxide nanocomposite based DLLME dSPE approach

Öz

This study aimed to develop and validate a sensitive, environmentally friendly analytical method for the simultaneous quantification of donepezil and rivastigmine in human plasma to support therapeutic drug monitoring (TDM). The approach combined a novel dispersive solid-phase extraction (dSPE) utilizing a purposely synthesized SBA-15/graphene oxide (GO) nanocomposite characterized by FT-IR, SEM, and TGA with dispersive liquid-liquid microextraction (DLLME), followed by HPLC-UV detection. Critical parameters affecting extraction efficiency, including sorbent amount, desorption/disperser solvent, sample pH, equilibration time, and ionic strength, were systematically optimized, and the method was validated according to ICH M10 and FDA (2018) guidelines. The optimized SBA-15/GO nanocomposite displayed a highly ordered mesoporous structure with an average pore diameter of 66.77 nm, with optimal extraction achieved using 0.001 g of sorbent, acetonitrile as the desorption/disperser solvent, a sample pH of 6.0, a 30-minute equilibration time, and no salt addition. The method exhibited excellent linearity over 0.15–200 µg/mL for donepezil and 0.01–50 µg/mL for rivastigmine (R² = 0.998 for both), with limits of detection of 0.04 and 0.002 µg/mL, respectively. Absolute extraction recoveries reached 83% for donepezil and 79% for rivastigmine, with enrichment factors of 10, while intra- and inter-assay precision (RSD) remained below 7.8% and 8.2%, respectively, and accuracy (RE) ranged from –6.0% to +7.1%. Clinical applicability was assessed by analyzing plasma samples from six medicated patients receiving either donepezil (5 or 10 mg/day) or rivastigmine (1.5 or 3 mg/day), with results confirming dose-proportional concentrations and demonstrating the method's suitability for routine TDM. Moreover, the procedure is rapid, with a total preparation time of less than 45 minutes, consumes minimal organic solvent (under 2 mL per sample), and adheres strictly to green analytical chemistry principles.

Anahtar Kelimeler

• Donepezil
• Rivastigmine
• Dispersive liquid-liquid microextraction coupled with dispersive solid-phase extraction (DLLME-dSPE)
• SBA-15/GO nanocomposite
• Therapeutic drug monitoring

Yeni bir SBA-15/Grafen Oksit Nanokompozit Tabanlı DLLME dSPE Yaklaşımı Kullanılarak İnsan Plazmasında Donepezil ve Rivastigminin Eş Zamanlı HPLC-UV Kantifikasyonu

Öz

This study aimed to develop and validate a sensitive, environmentally friendly analytical method for the simultaneous quantification of donepezil and rivastigmine in human plasma to support therapeutic drug monitoring (TDM). The approach combined a novel dispersive solid-phase extraction (dSPE) utilizing a purposely synthesized SBA-15/graphene oxide (GO) nanocomposite characterized by FT-IR, SEM, and TGA with dispersive liquid-liquid microextraction (DLLME), followed by HPLC-UV detection. Critical parameters affecting extraction efficiency, including sorbent amount, desorption/disperser solvent, sample pH, equilibration time, and ionic strength, were systematically optimized, and the method was validated according to ICH M10 and FDA (2018) guidelines. The optimized SBA-15/GO nanocomposite displayed a highly ordered mesoporous structure with an average pore diameter of 66.77 nm, with optimal extraction achieved using 0.001 g of sorbent, acetonitrile as the desorption/disperser solvent, a sample pH of 6.0, a 30-minute equilibration time, and no salt addition. The method exhibited excellent linearity over 0.15–200 µg/mL for donepezil and 0.01–50 µg/mL for rivastigmine (R² = 0.998 for both), with limits of detection of 0.04 and 0.002 µg/mL, respectively. Absolute extraction recoveries reached 83% for donepezil and 79% for rivastigmine, with enrichment factors of 10, while intra- and inter-assay precision (RSD) remained below 7.8% and 8.2%, respectively, and accuracy (RE) ranged from –6.0% to +7.1%. Clinical applicability was assessed by analyzing plasma samples from six medicated patients receiving either donepezil (5 or 10 mg/day) or rivastigmine (1.5 or 3 mg/day), with results confirming dose-proportional concentrations and demonstrating the method's suitability for routine TDM. Moreover, the procedure is rapid, with a total preparation time of less than 45 minutes, consumes minimal organic solvent (under 2 mL per sample), and adheres strictly to green analytical chemistry principles. ۲٬۰۱۷ Bu çalışma, terapötik ilaç izleme (TDM) çalışmalarını desteklemek amacıyla insan plazmasında donepezil ve rivastigminin eş zamanlı kantifikasyonu için hassas, çevre dostu bir analitik yöntem geliştirmeyi ve doğrulamayı amaçlamıştır. Yaklaşım, FT-IR, SEM ve TGA ile karakterize edilen özel olarak sentezlenmiş bir SBA-15/grafen oksit (GO) nanokompoziti kullanan yeni bir dağılımlı katı faz ekstraksiyonunu (dSPE) dağılımlı sıvı-sıvı mikroekstraksiyonu (DLLME) ile birleştirmiş ve ardından HPLC-UV dedektörü ile analiz etmiştir. Ekstraksiyon verimliliğini etkileyen kritik parametreler, sorbent miktarı, desorpsiyon/dağıtıcı çözücü, numune pH'ı, denge süresi ve iyonik kuvvet dahil olmak üzere sistematik olarak optimize edilmiş ve yöntem ICH M10 ve FDA (2018) kılavuzlarına göre doğrulanmıştır. Optimize edilmiş SBA-15/GO nanokompoziti, ortalama gözenek çapı 66,77 nm olan oldukça düzenli bir mezogözenekli yapı sergiledi; optimum ekstraksiyon, 0,001 g sorbent, desorpsiyon/dağıtıcı çözücü olarak asetonitril, 6,0'lık bir numune pH'ı, 30 dakikalık dengeleme süresi ve tuz ilavesi olmaksızın elde edildi. Yöntem, donepezil için 0,15–200 µg/mL ve rivastigmin için 0,01–50 µg/mL aralığında mükemmel doğrusallık gösterdi (her ikisi için de R² = 0,998), tespit limitleri sırasıyla 0,04 ve 0,002 µg/mL idi. Donepezil için mutlak ekstraksiyon geri kazanımı %83'e, rivastigmin için ise %79'a ulaşmıştır; zenginleştirme faktörleri 10 iken, intra- ve inter-analiz hassasiyeti (RSD) sırasıyla %7,8 ve %8,2'nin altında kalmış ve doğruluk (RE) -%6,0 ile +%7,1 arasında değişmiştir. Klinik uygulanabilirlik, donepezil (5 veya 10 mg/gün) veya rivastigmin (1,5 veya 3 mg/gün) alan altı ilaç kullanan hastadan alınan plazma örneklerinin analiz edilmesiyle değerlendirilmiş ve sonuçlar dozla orantılı konsantrasyonları doğrulayarak yöntemin rutin TDM için uygunluğunu göstermiştir. Dahası, işlem hızlıdır, toplam hazırlık süresi 45 dakikadan azdır, minimum organik çözücü tüketir (örnek başına 2 mL'den az) ve yeşil analitik kimya prensiplerine sıkı sıkıya bağlıdır.

Anahtar Kelimeler

• Donepezil
• Rivastigmin
• Dağıtıcı sıvı-sıvı mikroekstraksiyonun dağıtıcı katı faz ekstraksiyonu ile birleştirilmesi (DLLME-dSPE)
• SBA-15/GO nanokompoziti
• Terapötik ilaç izleme

Kaynakça

1. Alzheimer's Association, 2021 Alzheimer's disease facts and figures, Alzheimers Dement, 17, 2021, 327-406.
2. J.L. Cummings, R.S. Isaacson, F.A. Schmitt, D.M. Velting, A practical algorithm for managing Alzheimer's disease: What, when, and why?, Ann Clin Transl Neurol, 2, 2015, 307-323.
3. J.S. Birks, Cholinesterase inhibitors for Alzheimer's disease, Cochrane Database Syst Rev, 2006, CD005593.
4. H. Kaduszkiewicz, T. Zimmermann, H.P. Beck-Bornholdt, H. van den Bussche, Cholinesterase inhibitors for patients with Alzheimer's disease: Systematic review of randomised clinical trials, BMJ, 331, 2005, 321-327.
5. R. Cacabelos, Pharmacogenetic basis for therapeutic drug monitoring of cholinesterase inhibitors, Expert Opin Drug Metab Toxicol, 5, 2009, 1523-1542.
6. G. Hefner, A. Brueckner, C. Hiemke, A. Fellgiebel, Therapeutic drug monitoring for patients with Alzheimer dementia to improve treatment with donepezil, Ther Drug Monit, 37, 2015, 353-361.
7. C. Hiemke, N. Bergemann, H.W. Clement, A. Conca, J. Deckert, K. Domschke, Consensus guidelines for therapeutic drug monitoring in neuropsychopharmacology: Update 2017, Pharmacopsychiatry, 51, 2018, 9-62.
8. M.H.F. Fischer, I.C. Zibrandtsen, P. Johannsen, V. Siersma, J.B. Rasmussen, J.B. Larsen, P. Høgh, Therapeutic drug monitoring for dose optimization in Alzheimer's disease and in dementia with Lewy bodies: A randomized single-blinded clinical trial, J Alzheimers Dis Rep, 8, 2024, 1516-1528.

9. N. Yasui-Furukori, R. Furuya, T. Takahata, T. Tateishi, Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection, J Chromatogr B, 768, 2002, 261-265.
10. G. Zhang, Y. Zhang, C. Zhang, Y. Li, X. Wang, Simultaneous determination of donepezil, rivastigmine and their metabolites in human plasma by LC-MS/MS, J Pharm Biomed Anal, 138, 2017, 197-206.
11. DrugBank, Donepezil, Accession DB00843, 2026, https://go.drugbank.com/drugs/DB00843
12. DrugBank, Rivastigmine, Accession DB00989, 2026, https://go.drugbank.com/drugs/DB00989
13. K. Arumugam, M.R. Chamallamudi, S.R. Mallayasamy, R. Mullangi, S. Ganesan, L. Jamadar, High performance liquid chromatographic fluorescence detection method for the quantification of rivastigmine in rat plasma and brain: Application to preclinical pharmacokinetic studies in rats, J Young Pharm, 3, 2011, 14-21.
14. Y.R. Wang, Y.H. Yang, C.Y. Lu, S.J. Lin, S.H. Chen, Trace analysis of acetylcholinesterase inhibitors with antipsychotic drugs for Alzheimer's disease by capillary electrophoresis with on-column field-amplified sample injection, Anal Bioanal Chem, 405, 2013, 3233-3242.
15. A. Andrade-Eiroa, M. Canle, V. Leroy-Cancellieri, V. Cerdà, Solid-phase extraction of organic compounds: A critical review (Part I), TrAC Trends Anal Chem, 80, 2016, 641-654.
16. C. Polson, P. Sarkar, B. Incledon, V. Raguvaran, R. Grant, Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography–tandem mass spectrometry, J Chromatogr B, 785, 2003, 263-275.
17. A. Kalyniukova, J. Balogh, J. Jeko, Z. Cziaky, Application of liquid–liquid microextraction for the effective separation and simultaneous determination of 11 pharmaceuticals in wastewater samples using HPLC–MS/MS, J Sep Sci, 41, 2018, 2870-2877.
18. M.C. Hennion, Solid-phase extraction: Method development, sorbents, and coupling with liquid chromatography, J Chromatogr A, 856, 1999, 3-54.
19. M. Anastassiades, S.J. Lehotay, D. Štajnbahr, F.J. Schenck, Fast and easy multiresidue method employing acetonitrile extraction/partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce, J AOAC Int, 86, 2003, 412-431.
20. A. Zgola-Grzeskowiak, T. Grzeskowiak, Dispersive liquid–liquid microextraction, TrAC Trends Anal Chem, 30, 2011, 1382-1399.
21. O. Zuloaga, M. Olivares, P. Navarro, A. Vallejo, A. Prieto, Dispersive liquid–liquid microextraction: Trends in the analysis of biological samples, Bioanalysis, 7, 2015, 2211-2225.
22. I. Kohler, J. Schappler, T. Sierro, S. Rudaz, Dispersive liquid–liquid microextraction combined with capillary electrophoresis and time-of-flight mass spectrometry for urine analysis, J Pharm Biomed Anal, 73, 2013, 82-89.
23. R. Fazeli-Bakhtiyari, M.H. Sorouraddin, M.A. Farajzadeh, A. Jouyban, Detection limit enhancement of antiarrhythmic drugs in human plasma using capillary electrophoresis with dispersive liquid–liquid microextraction and field-amplified sample stacking method, Bioanalysis, 7, 2015, 21-37.
24. M. Zamani-Kalajahi, R. Fazeli-Bakhtiyari, M. Amiri, A. Golmohammadi, A. Afrasiabi, M. Khoubnasabjafari, Dispersive liquid–liquid microextraction based on solidification of floating organic droplet followed by spectrofluorimetry for determination of carvedilol in human plasma, Bioanalysis, 5, 2013, 437-448.
25. S. Soltani, A.M. Ramezani, N. Soltani, A. Jouyban, Analysis of losartan and carvedilol in urine and plasma samples using a dispersive liquid–liquid microextraction isocratic HPLC-UV method, Bioanalysis, 4, 2012, 2805-2821.
26. J.S. Beck, J.C. Vartuli, W.J. Roth, M.E. Leonowicz, C.T. Kresge, K.D. Schmitt, A new family of mesoporous molecular sieves prepared with liquid crystal templates, J Am Chem Soc, 114, 1992, 10834-10843.
27. A. Walcarius, E. Sibottier, M. Etienne, J. Ghanbaja, Electrochemically assisted self-assembly of mesoporous silica thin films, Nat Mater, 6, 2007, 602-608.
28. A. Badiei, L. Bonneviot, N. Crowther, G.M. Ziarani, Surface tailoring control in micelle templated silica, J Organomet Chem, 691, 2006, 5923-5931.
29. D.R. Dreyer, S. Park, C.W. Bielawski, R.S. Ruoff, The chemistry of graphene oxide, Chem Soc Rev, 39, 2010, 228-240.
30. O.C. Compton, S.T. Nguyen, Graphene oxide, highly reduced graphene oxide, and graphene: Versatile building blocks for carbon-based materials, Small, 6, 2010, 711-723.
31. A. Mirabi, A. Shokuhi Rad, Z. Khanjari, M. Moradian, Preparation of SBA-15/graphene oxide nanocomposites for preconcentration and determination of trace amounts of rutoside in blood plasma and urine, Sens Actuators B Chem, 249, 2017, 187-194.
32. Z. Aladaghlo, A. Fakhari Zavareh, S.I. Alavioon, M. Dabiri, A mesoporous nanosorbent composed of silica, graphene, and palladium (II) for ultrasound-assisted dispersive solid-phase extraction of organophosphorus pesticides prior to their quantitation by ion mobility spectrometry, Microchim Acta, 187, 2020, 187-209.
33. ICH, ICH guideline M10 on bioanalytical method validation, European Medicines Agency, 2019.
34. U.S. Food and Drug Administration, Bioanalytical method validation: Guidance for industry, Silver Spring, MD: FDA, 2018.
35. W.S. Hummers, R.E. Offeman, Preparation of graphitic oxide, J Am Chem Soc, 80, 1958, 1339.
36. T.X. Bui, H. Choi, Adsorptive removal of selected pharmaceuticals by mesoporous silica SBA-15, J Hazard Mater, 168, 2009, 602-608.
37. T.X. Bui, S.Y. Kang, S.H. Lee, H. Choi, Organically functionalized mesoporous SBA-15 as sorbents for removal of selected pharmaceuticals from water, J Hazard Mater, 193, 2011, 156-163.
38. S. Yildirim, N.U. Colak, A. Yasar, Application of dispersive liquid–liquid microextraction for the determination of donepezil in urine by HPLC using a core–shell column, J Liq Chromatogr Relat Technol, 41, 2017, 120-128.
39. A. Pilotto, M. Franceschi, G. D'Onofrio, A. Bizzarro, F. Mangialasche, L. Cascavilla, Effect of a CYP2D6 polymorphism on the efficacy of donepezil in patients with Alzheimer disease, Neurology, 73, 2009, 761-767.
40. A. Gałuszka, Z.M. Migaszewski, P. Konieczka, J. Namieśnik, Analytical Eco-Scale for assessing the greenness of analytical procedures, TrAC Trends Anal Chem, 37, 2012, 61-72.