Aplicação do método em amostras reais

O processo de validação engloba a aplicação do método desenvolvido em amostras reais, demonstrado a sua eficácia em situações reais. Como tal, antes de proceder a este estudo e a fim de recolher amostras de indivíduos sob tratamento com antiepiléticos o presente trabalho

108

foi submetido à Comissão de Ética da Universidade da Beira Interior, sendo este aprovado (Processo n.º CE-UBI-Pj-2017-013).

Foram recolhidas por spitting amostras de fluido oral de voluntários que se encontravam em terapêutica ambulatória com CBZ, FNT ou FBT. A recolha foi feita em Farmácias do Grupo Holon-Covilhã. Estas amostras foram conservadas a -20ºC até à sua análise. Após secagem da respetiva mancha de acordo com o procedimento anteriormente descrito procedeu-se à sua extração e análise cromatográfica. Na tabela 17 encontram-se descritas as concentrações obtidas, identificando o antiepilético detetado.

A modo de exemplo na figura 16 encontra-se representado um cromatograma relativo a amostra número 1.

Tabela 17. Listagem de compostos identificados nas amostras reais e respetiva quantificação.

Amostra Antiepilético Concentração (µg/mL)

1 Fenobarbital 3,49

2 Carbamazepina 1,34

3 Carbamazepina 0,53

4 Fenitoína 1,57

5 Fenitoína 1,79

109

Considerações finais

1. Apresenta-se um método analítico de fácil e rápida execução baseado na utilização de DSS como técnica de preparação da amostra para a determinação de fármacos anticonvulsivos em fluido oral, mais concretamente CBZ (e o seu metabolito ativo E- CBZ), FNT e FNB. A análise foi realizada por HPLC acoplada a um detetor DAD.

2. Os principais parâmetros suscetíveis de afetar o processo de extração foram previamente otimizados com o objetivo de maximizar a recuperação e assim obter baixos limites de deteção e quantificação. Assim sendo foram obtidos a limites de deteção e quantificação baixos de 0,05 e 0,1 µg/mL, respetivamente e recuperações médias de 50% de todos os compostos de estudo.

3. O procedimento de extração desenvolvido é simples, sensível e eficaz, utilizando um volume de amostra baixo (50 µL).

4. O método demonstrou-se seletivo, preciso, exato e linear, estando de acordo com os critérios internacionais de validação de métodos analíticos.

5. O método foi aplicado com sucesso a amostras reais, pelo que o método proposto demonstrou ser uma excelente ferramenta para o realizar o seguimento farmacoterapêutico de medicamentos anticonvulsivos.

6. Salienta-se ainda que o método descrito é o primeiro estudo que permite identificar e quantificar estes compostos em amostras de fluido oral com recurso aos DSS.

110

Bibliografia

[1] P. Abu-rabie and N. Spooner, “Dried matrix spot direct analysis : evaluating the robustness of a direct elution technique for use in quantitative bioanalysis,” Bioanalysis, vol. 3, no. 24, pp. 2769–2781, 2011.

[2] J. S. Zimmer, C. D. Christianson, C. J. Johnson, and S. R. Needham, “Recent advances in the bioanalytical applications of dried matrix spotting for the analysis of drugs and their metabolites,” Bioanalysis, vol. 5, no. 20, pp. 2581–2588, 2013.

[3] A. Abdel-Rehim and M. Abdel-Rehim, “Dried saliva spot as a sampling technique for saliva samples,” Biomed. Chromatogr., vol. 28, no. 6, pp. 875–877, 2014.

[4] M. Numako et al., “Dried Saliva Spot (DSS) as a Convenient and Reliable Sampling for Bioanalysis: An Application for the Diagnosis of Diabetes Mellitus,” Anal. Chem., vol. 88, no. 1, pp. 635–639, 2016.

[5] W. H. Hannon and B. L. Therrell, “HISTORY, APPLICATIONS, AND HEALTHCARE,” in Dried Blood Spots: Applications and Techniques, W. Li and M. S. Lee, Eds. Wiley, 2014. [6] J. C. Atkinson et al., “Guidelines for Saliva Nomenclature and Collection,” Ann. N. Y.

Acad. Sci., vol. 694, pp. xi–xii, 1993.

[7] S. P. Humphrey and R. T. Williamson, “A review of saliva: Normal composition, flow, and function,” J. Prosthet. Dent., vol. 85, no. 2, pp. 162–169, 2001.

[8] J. W. Paxton and B. Whiting, “PHENYTOIN CONCENTRATIONS IN MIXED , PAROTID AND SUBMANDIBULAR SALIVA AND SERUM MEASURED BY RADIOIMMUNOASSAY,” Br. J. Clin. Pharmacol., vol. 4, pp. 185–191, 1977.

[9] J. K. M. Aps and L. C. Martens, “Review: The physiology of saliva and transfer of drugs into saliva,” Forensic Sci. Int., vol. 150, no. 2–3, pp. 119–131, 2005.

[10] E. Gallardo and J. A. Queiroz, “The role of alternative specimens in toxicological analysis,” Biomed. Chromatogr., vol. 22, no. 8, pp. 795–821, 2008.

[11] E. Gallardo, M. Barroso, and J. a Queiroz, “Current technologies and considerations for drug bioanalysis in oral fluid.,” Bioanalysis, vol. 1, no. 3, pp. 637–67, 2009.

[12] R. E. Choo and M. A. Huestis, “Oral fluid as a diagnostic tool,” Clin. Chem. Lab. Med., vol. 42, no. 11, pp. 1273–1287, 2004.

[13] S. Basalingappa, A. Sharma, and S. Amarnath, “Basic Concepts of Therapeutic Drug Monitoring,” Int. J. Curr. Pharm. Rev. Res., vol. 5, no. 4, pp. 70–75, 2014.

[14] M. Hallworth, “Therapeutic Drug Monitoring,” in Clarke’s Analysis and Poisons, 4th ed., A. C. Moffat, M. D. Osselton, and B. Widdop, Eds. Pharmaceutical Press, 2011, p. 59-.

[15] R. Ghiculescu, “Therapeutic drug monitoring: which drugs, why, when and how to do it,” Aust. Prescr., vol. 31, no. 2, 2008.

[16] J.-S. Kang and M.-H. Lee, “Overview of Therapeutic Drug Monitoring,” Korean J. Intern. Med., vol. 24, no. 1, pp. 1–10, 2009.

111 New Therapeutic Drug Monitoring Strategies: A Comprehensive Review, Expert Opin Drug Metab Toxicol. vol. 0, no. 0. Taylor & Francis, 2017.

[18] T. L. Lemke, D. A. Williams, V. F. Roche, and S. W. Zito, “Antiseizure Agents,” in Foye’s Principles of Medicinal Chemistry, 6th Edition., 2008, pp. 521–546.

[19] J. O. McNamara, “PHARMACOTHERAPY OF THE EPILEPSIES,” in Goodman & Gilman’s - The Pharmacological Basis of Therapeutics, 2011, pp. 501–525.

[20] S. D. Shorvon, “The etiologic classification of epilepsy,” Epilepsia, vol. 52, no. 6, pp. 1052–1057, 2011.

[21] A. T. Berg et al., “Revised terminology and concepts for organization of seizures and epilepsies: Report of the ILAE Commission on Classification and Terminology, 2005- 2009,” Epilepsia, Vol.51, no. 4, pp.676-85, 2010.

[22] National Institute for Health and Care Excellence (NICE), “Epilepsies : diagnosis and management”, 2016. [Online]. Available: https://www.nice.org.uk/guidance/cg137 [Accessed: 12-Jan-2017].

[23] P. A. Datar, “Quantitative bioanalytical and analytical method development of dibenzazepine derivative , carbamazepine : A review,” J. Pharm. Anal., vol. 5, no. 4, pp. 213–222, 2015.

[24] Pubchem, “Carbamazepina,” 2017. [Online]. Available: https://pubchem.ncbi.nlm.nih.gov/compound/carbamazepine#section=Top.

[Accessed: 12-Jan-2017].

[25] Infarmed, “Infomed.” [Online]. Available:

http://app7.infarmed.pt/infomed/pesquisa.php. [Accessed: 11-Jul-2017].

[26] B. W. Abou-khalil, “Antiepileptic Drugs,” Continuum (N. Y)., no. February, pp. 132– 156, 2016.

[27] S. K. Bedada, R. Appani, and P. K. Boga, “Effect of Piperine on the Metabolism and Pharmacokinetics of Carbamazepine in Healthy Volunteers,” Drug Res. (Stuttg)., vol. 67, no. 1, pp. 46–51, 2016.

[28] F. A. Ibrahim, A. F. El-Yazbi, M. A. Barary, and M. M. Wagih, “Sensitive inexpensive HPLC determination of four antiepileptic drugs in human plasma : application to PK studies,” Bioanalysis, vol. 8, no. 21, pp. 2219–2234, 2016.

[29] E. Spina, F. Pisani, and J. De Leon, “CLINICALLY SIGNIFICANT PHARMACOKINETIC DRUG INTERACTIONS OF ANTIEPILEPTIC DRUGS WITH NEW ANTIDEPRESSANTS AND NEW ANTIPSYCHOTICS,” Pharmacol. Res., vol. 106, pp. 72–86, 2016.

[30] J. W. Paxton and R. A. Donald, “Concentrations and kinetics of carbamazepine in whole saliva, parotid saliva, serum ultrafiltrate, and serum,” Clin. Pharmacol. Ther., pp. 695–702, 1980.

[31] R. Dwivedi, M. Singh, T. Kaleekal, Y. K. Gupta, and M. Tripathi, “Concentration of antiepileptic drugs in persons with epilepsy: a comparative study in serum and saliva.,” Int. J. Neurosci., vol. 7454, pp. 1–7, 2015.

112

Absolute Oral Bioavailability Determined by Use of a Stable Isotope in Patients With Epilepsy,” Ther. Drug Monit., vol. 33, no. 1, pp. 56–63, 2011.

[33] Pubchem, “Phenytoin,” 2005. [Online]. Available:

https://pubchem.ncbi.nlm.nih.gov/compound/phenytoin#section=Top. [Accessed: 12- Jan-2017].

[34] Infarmed and Ministério da Saúde, Monografias - Farmacopeia Portuguesa, 9.0. 2008. [35] Pubchem, “Phenobarbital,” 2005. [Online]. Available:

https://pubchem.ncbi.nlm.nih.gov/compound/phenobarbital#section=Top. [Accessed: 12-Jan-2017].

[36] G. M. Pacifici, “Clinical Pharmacology of Phenobarbital in Neonates: Effects, Metabolism and Pharmacokinetics,” Curr. Pediatr. Rev., vol. 12, no. 1, pp. 48–54, 2016.

[37] D. Fritch, K. Blum, S. Nonnemacher, K. Kardos, A. R. Buchhalter, and E. J. Cone, “Barbiturate detection in oral fluid, plasma, and urine.,” Ther. Drug Monit., vol. 33, no. 1, pp. 72–79, 2011.

[38] Z. Yasiry and S. D. Shorvon, “How phenobarbital revolutionized epilepsy therapy: the story of phenobarbital therapy in epilepsy in the last 100 years.,” Epilepsia, vol. 53 Suppl 8, pp. 26–39, 2012.

[39] Food and Drug Administration (FDA), “Guidance for Industry: Bioanalytical method validation.,” U.S. Department of Health and Human Services, 2013. [Online]. Available: https://www.fda.gov/downloads/Drugs/Guidances/ucm368107.pdf.

[40] EMA, “Guideline on bioanalytical method validation,” 2012. [Online]. Available: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011 /08/WC500109686.pdf.

[41] M. C. Hill, “Methods and Guidelines for Effective Model Calibration,” Denver, Colorado, 1998.

[42] S. Đorđević, V. Kilibarda, and T. Stojanović, “Determination of carbamazepine in serum and saliva samples by high performance liquid chromatography with ultraviolet detection,” Vojn. Pregl, vol. 66, no. 5, pp. 347–352, 2009.

[43] E. Greiner-sosanko, S. Giannoutsos, D. R. Lower, M. A. Virji, and M. D. Krasowski, “Drug Monitoring : Simultaneous Analysis of Lamotrigine , Oxcarbazepine , 10- Hydroxycarbazepine , and Zonisamide by HPLC – UV and a Rapid GC Method Using a Nitrogen-Phosphorus Detector for Levetiracetam,” J. Chromatogr. Sci., vol. 45, no. October, pp. 616–622, 2007.

[44] M. Vosough, S. Ghafghazi, and M. Sabetkasaei, “Chemometrics enhanced HPLC-DAD performance for rapid quantification of carbamazepine and phenobarbital in human serum samples,” Talanta, vol. 119, pp. 17–23, 2014.

[45] A. Ferreira, M. Rodrigues, A. Falcão, and G. Alves, “A Rapid and Sensitive HPLC-DAD Assay to Quantify Lamotrigine, Phenytoin and Its Main Metabolite in Samples of Cultured HepaRG Cells,” J. Chromatogr. Sci., vol. 54, no. 8, pp. 1352–1358, 2016.

113 [46] B. Anilanmert et al., “Simultaneous analysis method for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin in urine, using C18 poroshell column,” J. Chromatogr. B Anal. Technol. Biomed. Life Sci., vol. 1022, pp. 230–241, 2016.

[47] A. Song, “Determination of 13 Organic Toxicants in Human Blood by Liquid – Liquid Extraction Coupling High-Performance Liquid Chromatography Tandem Mass Spectrometry,” Anal. Sci., vol. 32, no. 6, pp. 645-652, 2016.

[48] L. Yin et al., “Simultaneous determination of ten antiepileptic drugs in human plasma by liquid chromatography and tandem mass spectrometry with positive/negative ion- switching electrospray ionization and its application in therapeutic drug monitoring,” J. Sep. Sci., vol. 39, no. 5, pp. 964–972, 2016.

[49] N. M. Shah, A. F. Hawwa, J. S. Millership, P. S. Collier, and J. C. McElnay, “A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots,” J. Chromatogr. B Anal. Technol. Biomed. Life Sci., vol. 923–924, pp. 65–73, 2013.

115