J. l. e T.V. são suportados pelo Instituto Nacional de alergia e doenças infecciosas (NIAID) dos institutos nacionais de saúde (R01 AI111965). O conteúdo é exclusivamente da responsabilidade dos autores e não representam necessariamente as opiniões oficiais do Instituto Nacional de alergia e doenças infecciosas ou o National Institutes of Health. MC é um pesquisador australiano NHMRC Principal. J. L. é um australiano de saúde nacional e Conselho de pesquisa médica (NHMRC) Senior Research Fellow e T.V. é um australiano NHMRC indústria carreira desenvolvimento nível 2 Research Fellow. E.K.S é um nomeado embaixador Young 2017 para ASM (sociedade americana de microbiologia) e é suportado pelo prêmio de pós-graduação australiana. Partes deste trabalho foi apresentado no 12th Australiasian conferência sobre fibrose cística em Melbourne (5-8th de agosto de 2017).

aprovação ética foi obtida da Monash University humano pesquisa ética Comissão (MUHREC).
 1. aplicação do ensaio: coleta de amostra de paciente <ol><li>registrar o tempo quando o paciente toma sua dose padrão de 150 mg ivacaftor ou /lumacaftor de 125 mg de ivacaftor 200 mg.</li>
	<li>Nota para baixo o tempo exato quando a amostra de sangue do paciente é recolhida. 
	Nota: Recomendamos que a coleta de amostras de 4-5 sobre um curso de tempo de 24 h. Se a coleta de única amostra é possível, o ponto do tempo indicado é 2.5-4 h post ivacaftor ou ivacaftor-lumacaftor combinação de dosagem como C máx concentrações no estado estacionário será realizáveis após &#62; 5 dias de tratamento consecutivo.</li>
	<li>Coletar 4-5 mL de sangue em tubos de azuis não tratados comercialmente disponíveis.</li>
	<li>Após a coleta do sangue total, permitem que o sangue a coagular, deixando-a intacta à temperatura ambiente. 
	Nota: Isto normalmente leva 15-30 min.</li>
	<li>Remover o coágulo por centrifugação a 1.000-2.000 x g durante 10 minutos a 4 ° C em uma centrífuga refrigerada. Designar o sobrenadante resultante como plasma.</li>
	<li>Após centrifugação, imediatamente transferir o sobrenadante (plasma) em um tubo limpo polipropileno usando uma pipeta Pasteur. Rótulo o plasma do tubo com a identificação do paciente (por exemplo, 1 paciente), a droga administrada (por exemplo, combinação de ivacaftor-lumacaftor) e tempo de amostra do paciente foi coletado (por exemplo, pós-dosagem de 2,5 h).</li>
	<li>Amostras de manter a 2-8 ° C durante a manipulação. Uma vez que o tratamento é concluído, armazenar as amostras em &#8211; 20 ° C.</li>
	<li>, Se necessário, enviamos amostras com um mensageiro aprovado ao Instituto analisando. 
	Nota: As amostras estão a ser transferidos em gelo seco até a chegada e devem ser mantidas em &#8211; 80° C até análise.</li>
</ol> 2. Preparação e processamento de amostras efectuadas e normas Nota: Plasma de doadores saudáveis ingênuo para terapia ivacaftor/lumacaftor foi obtido da Cruz Vermelha australiana. Para garantir a integridade, todas as amostras/padrões devem ser mantidos a 2-8 ° C durante a colheita e o processamento. Uma vez que o tratamento é concluído, armazenar as amostras em &#8211; 20 ° C.
<ol><li>Pesar IVA, IVA-carboxilato, hidroximetil-IVA e LUMA, que servirá como referência para as normas internas.</li>
	<li>Preparar duas soluções estoque independentes de cada analito (IVA, M1, M6 e LUMA) em metanol de grau de LC-MS a 100 µ g/mL e 10 µ g/ml. 
	Nota: Compostos expirará após 10 dias sobre a bancada.</li>
	<li>Recém preparar padrões de calibração de LC-MS antes de cada execução analítica por diluição dos padrões de calibração Soluções conservadas em estoque em plasma humano para atingir concentrações na sequência: 0,01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5,0 e 10,0 µ g/mL. Estas amostras são referidas como padrões.</li>
	<li>De precipitar proteínas, preparar 0,1% de ácido fórmico (FA) em acetonitrila (ACN, grau de LC-MS) e deixar na geladeira até ser necessário.</li>
</ol> 3. Pré-tratamento de amostras efectuadas e normas <ol><li>permitir que ambas as amostras de plasma do paciente e em branco equilibrar a temperatura ambiente.</li>
	<li>Vortex para misturar cada amostra de plasma por 15 s por exemplo.</li>
	<li>Transferir uma alíquota de 100 µ l de qualquer plasma em branco para os padrões ou as amostras para análise em um tubo de polipropileno microcentrifuga 1,5 mL.</li>
	<li>Acrescentar a IVA de padrão interna em cada um dos tubos padrão (por exemplo, 0,01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 e 10,0 µ g/mL para cobrir o intervalo de concentração potencial), feche a tampa e vórtice para 2-3 s. nota que a norma interna é apenas adicionado ao plasma em branco e sem padrão interno é adicionado das amostras.</li>
	<li>Repita o passo 3.4. para os outros padrões internos, M1, M6 e LUMA.</li>
	<li>Spin-down cada tubo brevemente para assegurar que não há nenhum gotículas na tampa.</li>
	<li>Adicionar 200 µ l da mistura de 0,1% de FA na ACN em cada tubo de precipitar proteínas plasmáticas.</li>
	<li>Vortex a mistura vigorosamente por cerca de 15 s.</li>
	<li>Permitir que tubos repousar durante 10 min na geladeira.</li>
	<li>Centrifugar a 10.000 x g durante 10 minutos a 4 ° C, se possível.</li>
	<li>Filtrar num filtro de seringa de 13 mm em um frasco HPLC de 1,5 mL uma alíquota de 200 µ l do sobrenadante.</li>
	<li>Transferir 100 µ l do sobrenadante dentro do frasco de LC-MS para a análise de HPLC-MS.</li>
</ol> 4. Análise de HPLC-MS Nota: HPLC-MS a análise foi realizada em um sistema de LC-MS, juntamente com o espectrômetro de massa triplo quadrupolo (tabela 1).
<ol><li>Transformar o sistema de LC-MS, coloque a bandeja com as amostras no autoamostrador.</li>
	<li>Unir a coluna a uma coluna de guarda e se conectar ao sistema de LC-MS.</li>
	<li>Anexar as duas garrafas de fases móveis (frasco r: 100% do Grupo ACN; garrafa b: 0,1% de ácido fórmico na água) e equilibrar o sistema de LC-MS.</li>
	<li>Incorporar os seguintes parâmetros do protocolo de LC-MS.
	<ol><li>Dividir o fluxo de fase móvel antes de entrar o espectrômetro de massa na proporção de 2:1 (resíduos: entrada de MS).</li>
		<li>Realizar uma gradiente eluição com um caudal de 0,5 mL/min usando uma fase móvel composta de 100% do Grupo ACN e 0,1% ácido fórmico em água (inicio em 40: 60, v/v). Note-se que a porcentagem de volume de fase móvel muda ao longo da análise.</li>
		<li>Operar o espectrômetro de massa em um modo de ionização electrospray positivo.</li>
		<li>Certifique-se de que as configurações de LC-MS são os seguintes: tensão de pulverizador de íon 4,5 kV, energia de colisão 295.9 V, gás de nebulização: nitrogênio em 3 L/min; gás de colisão: argônio; secagem gás vazão 20 L/min, lente tensão Q3:-22 V, desolvation temperatura 250 ° C, com um bloco de calor temperatura de 400 ° C.</li>
		<li>Volume de injectar 10 µ l por exemplo.</li>
		<li>Detectar analitos usando múltiplas reação monitoramento (MRM). Monitorar as transições do íon de m/z 392.49 &#8594; 393, m/z 408.49 &#8594; 409, m/z 422.47 &#8594; 423 e m/z 452.40 &#8594; 453 para IVA, IVA-M1, IVA-M6 e LUMA, respectivamente. 
		Nota: O método recém otimizado ainda tem que ser totalmente validado conforme normas FDA.</li>
	</ol></li></ol> 5. As curvas de calibração <ol><li>as curvas de calibração de construção o LC-MS antes de cada corrida analítica, usando a relação entre as proporções de área do pico de cada uma das substâncias a quatro analisar de padrão interno e as concentrações nominais padrão de calibração de ivacaftor (M1-IVA, IVA-M6 ou LUMA) dentro do programa de configurações de LC-MS. 
	Nota: As etapas exatas para a construção da curva de calibração é dependente do modelo de equipamento de LC-MS sendo usado. Essas informações podem estar disponíveis no manual do equipamento.</li>
	<li>Realizar a análise de regressão linear menos-quadrados por ponderação 1/C 2, de acordo com a recíproca das concentrações.</li>
</ol>

<ol>
	<li>Schneider, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-drug+plasma+protein+binding+interactions+of+ivacaftor.">Drug-drug plasma protein binding interactions of ivacaftor.</a> J Mol Recognit. 28 (6), 339-348 (2015).</li><li>Solomon, M., Leatte, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Treatments for Cystic fibrosis. , (2009).</li><li>O'Sullivan, B. P., Flume, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+approach+to+lung+disease+in+patients+with+cystic+fibrosis.">The clinical approach to lung disease in patients with cystic fibrosis.</a> Semin Respir Crit Care Med. 30 (5), 505-513 (2009).</li><li>Ramsey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CFTR+potentiator+in+patients+with+cystic+fibrosis+and+the+G551D+mutation.">A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.</a> N Engl J Med. 365 (18), 1663-1672 (2011).</li><li>Wainwright, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.</a> N Engl J Med. 373 (3), 220-231 (2015).</li><li>Hadida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide+(VX-770,+ivacaftor),+a+potent+and+orally+bioavailable+CFTR+potentiator.">Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator.</a> J Med Chem. 57 (23), 9776-9795 (2014).</li><li>FDA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FDA Advisory Commitee Briefing Material VERTEX-FDA Pulmonary-Allergy drugs advisory commitee. 98 , (2015).</li><li>Holmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=False+dawn+for+cystic+fibrosis+disease+modifiers?.">False dawn for cystic fibrosis disease modifiers?.</a> Nat Rev Drug Discov. 13 (10), 713-714 (2014).</li><li>Veit, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+gating+potentiators,+including+VX-770,+diminish+DeltaF508-CFTR+functional+expression.">Some gating potentiators, including VX-770, diminish DeltaF508-CFTR functional expression.</a> Sci Transl Med. 6 (246), 246ra297 (2014).</li><li>Cholon, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiator+ivacaftor+abrogates+pharmacological+correction+of+DeltaF508+CFTR+in+cystic+fibrosis.">Potentiator ivacaftor abrogates pharmacological correction of DeltaF508 CFTR in cystic fibrosis.</a> Sci Transl Med. 6 (246), 246ra297 (2014).</li><li>EMA, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment report ORKAMBI (ivacaftor/lumacaftor) European medicines agency. , (2015).</li><li>VERTEX. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertex prescribing infomation. , (2015).</li><li>Matthes, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+free+drug+concentration+prevents+inhibition+of+F508del+CFTR+functional+expression+by+the+potentiator+VX-770+(ivacaftor).">Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor).</a> Br J Pharmacol. 173 (3), 459-470 (2016).</li><li>Schneider, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+HPLC+and+LC-MS/MS+methods+for+the+analysis+of+ivacaftor,+its+major+metabolites+and+lumacaftor+in+plasma+and+sputum+of+cystic+fibrosis+patients+treated+with+ORKAMBI+or+KALYDECO.">Development of HPLC and LC-MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with ORKAMBI or KALYDECO.</a> J Chrom B Analyt Technol Biomed Life Sci. 1038, 57-62 (2016).</li><li>Su, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+LC-MS/MS+method+for+the+quantitation+of+cabozantinib+in+rat+plasma:+application+to+a+pharmacokinetic+study.">An LC-MS/MS method for the quantitation of cabozantinib in rat plasma: application to a pharmacokinetic study.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 985, 119-123 (2015).</li></ol>

Os autores não têm nada para divulgar.

Como relatado anteriormente, o nosso grupo tem para primeira vez desenvolvido e validado um método HPLC e LC-MS para a rápida detecção e quantificação de ivacaftor e seus principais metabolitos M1 hidroximetil-IVA (ativo) e IVA-carboxilato M6 (inativo); e lumacaftor no plasma e escarro de CF pacientes14. O ensaio relatado anteriormente pelo nosso grupo foi usado com sucesso para quantificar a concentração de LUMA, IVA, IVA-M1 e IVA-M6 no plasma e escarro de pacientes com FC submetidos à t...

Fibrose cística (CF) é uma doença genética comum, que envolve as glândulas exócrinas muco do pulmão, fígado, pâncreas e intestino causando insuficiência de múltiplos órgão progressiva, como um declínio na função pulmonar e insuficiência pancreática1, 2,3. Ivacaftor (IVA) é o primeiro Food and Drug Administration (FDA-EUA) e a Agência Europeia de medicamentos (EMA) aprovou a droga de potenciador do regulador (CFTR) de condutância de trans-membrana fibrose cística, com eficácia clínica evidenciada, produzindo uma significativa melhora na função pulmonar sobre placebo em um pequeno subconjunto de pacientes com FC tendo o G551D-CFTR [glicina (G) na posição 551 é substituída pelo ácido aspártico (D)] mutação missense (~ 4-5% da população CF)4,5. Esta administrada por via oral a droga aumenta o canal CFTR, abrindo, assim, aumentar o fluxo de íons cloreto e agindo sobre o defeito primário que leva para as manifestações clínicas da CF4,6. Infelizmente, o IVA em monoterapia não é eficaz em pacientes com a mutação mais comum de F508del homozigota [na exclusão de quadro do gene CFTR que resulta na perda de fenilalanina (F) na posição 508] que resulta em misfolded CFTR, o que é visto em ~ 50% de o CF população7,8.
Recentemente, o FDA concedeu aprovação para a combinação de IVA com o corrector CFTR lumacaftor de drogas. A estratégia inteligente de combinar um corrector CFTR (lumacaftor, LUMA) que resgata F508del-CFTR à superfície da célula com um modulador (IVA), que potencializa a atividade de canais CFTR, efetivamente expande a janela de tratamento para a maioria da população CF5 . Dúvidas sobre se essas drogas vão cumprir sua promessa como um número de relatos conflitantes surgiram dúvidas sobre sua eficácia clínica9,10elenco. Além disso, melhorias na função pulmonar foram apenas modesta (2.6-4% para a combinação de ivacaftor-lumacaftor) em comparação com o sucesso alcançado com IVA monoterapia em pacientes tendo um G551D mutação (10.6-12,5%)8. Potenciais interacções medicamentosas antagônica entre IVA e LUMA que potencialmente limitam a eficácia clínica da combinação de ivacaftor-lumacaftor vem de suas propriedades farmacocinéticas ideal menos de7,11. IVA é extensivamente metabolizada pelas enzimas de citocromo P450 (CYP), principalmente para um metabólito ativo hidroximetil-IVA (IVA-M1, M1) e uma forma inactiva IVA-carboxilato (IVA-M6, M6)7,12. O indutor de CYP3A4 LUMA, por outro lado, não é extensivamente metabolizado e é largamente excretado inalterado na fezes11. Como indutores de CYP3A4 induzem o metabolismo do citocromo, concentrações de ivacaftor (substrato de CYP3A4) poderiam ser reduzidas. Além disso, tanto o IVA e LUMA são moléculas muito hidrofóbicas e são ~ 99% ligado às proteínas plasmáticas, que limita significativamente a concentração de droga livre (ativa)1,13.
Coletivamente, esses fatores podem estar chegando juntos para limitar a eficácia clínica da combinação de ivacaftor-lumacaftor. Não se sabe se as concentrações plasmáticas ideais são alcançadas sob o regime de dosagem atual para combinação de ivacaftor-lumacaftor ou se o limiar terapêutico é mantido8. Atualmente, há uma escassez de informações sobre parâmetros farmacocinéticos, como o pico e concentrações plasmáticas de estado estacionário de ivacaftor ou ivacaftor-lumacaftor. Tendo em conta o metabolismo notável de ivacaftor e lumacaftor, monitoramento das relações de exposição-resposta é indispensável para alcançar regimes de dosagem ideal para terapia de ivacaftor ou ivacaftor-lumacaftor. Nosso grupo publicou recentemente o primeiro método HPLC/LC-MS para o monitoramento das relações de exposição-resposta de IVA e LUMA14. Não há técnicas alternativas de medir as concentrações de ivacaftor, seus metabólitos e lumacaftor foram relatadas até à data. Para permitir a análise do elevado-throughput de um coletivo maior do paciente e reduzir drasticamente o tempo de análise, o nosso grupo otimizou o método relatado com o uso de uma coluna de cromatografia de fase reversa de tamanho de poros menor e um sistema de solvente gradiente que reduz custos e tempos de corrida.

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:480px;">IVA (Cat#S114)&#160;</td>
			<td style="width:229px;">SelleckChem (USA).&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LUMA (Cat#S1565)&#160;</td>
			<td>SelleckChem (USA).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVA-carboxylate (Cat# 510242247CS)&#160;</td>
			<td>Clearsynth (Canada).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydroxymethyl-IVA (Cat# 510240849CS)&#160;</td>
			<td>Clearsynth (Canada).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol (MeOH, LC-MS grade),&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetonitrile (ACN, LC-MS grade)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formic acid (FA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triple-quadrupole Shimadzu 8030 LC-MS&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenomenex Kinetex (2.6 &#181;m C8 100 &#197;; 50 &#215; 2.1mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(KrudKatcher Ultrea HPLC In-Line Filter 0.5 m Depth Filter x 0.004inID).&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL polypropylene microcentrifuge tube (VWR).&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Centrifuge 5430</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">13-mm syringe filter (0.45 &#181;m nylon, GRACE, USA)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[Phenomenex VEREX, 9 mm, PP, 300 &#181;L, PTFE/Silicone septa].&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized lc-ms/ms method for the high-throughput analysis of clinical samples of ivacaftor, its major metabolites, and lumacaftor in biological fluids of cystic fibrosis patients

Defeitos no regulador de condutância trans-membrana de fibrose cística (CFTR) são a causa da fibrose cística (CF), uma doença com manifestações pulmonares fatais. Ivacaftor (IVA) e a combinação de ivacaftor-lumacaftor (LUMA) são duas novas drogas descoberta CF que modulam diretamente a atividade e o tráfico da proteína CFTR defeituosa. No entanto, ainda há uma falta de entendimento sobre os parâmetros farmacocinéticos/farmacodinâmicos e a farmacologia do ivacaftor e lumacaftor. A técnica de HPLC-MS para a análise simultânea das concentrações de hidroximetil-ivacaftor, ivacaftor, ivacaftor-carboxilato e lumacaftor em fluidos biológicos em pacientes recebendo a combinação padrão de ivacaftor ou ivacaftor-lumacaftor terapia já havia sido desenvolvido pelo nosso grupo e parcialmente validado para padrões do FDA. No entanto, para permitir a análise da elevado-produção de um maior número de amostras de doentes, nosso grupo otimizou o método relatado com o uso de uma coluna de cromatografia de fase reversa de tamanho de poro menor (2,6 µm, C8 100 Å; 50 x 2.1 mm) e um sistema de solvente gradiente ( 0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: realizada em 70% B; 2,7-2,8 min: 70-90% B; 2.8-lavagem de B 4,0 min: 90%; 4.0-4,1 min: 90-40% B; 4.1-6.0 min: realizada em 40% B) em vez de uma eluição isocrática. O objetivo deste estudo foi reduzir o tempo de análise de HPLC-MS por amostra dramaticamente de ~ 15 min para apenas 6 min, por exemplo, que é essencial para a análise de uma grande quantidade de amostras de doentes. Esse método conveniente será de considerável utilidade para estudos nas relações de exposição-resposta dessas drogas de CF de avanço.

Temos recentemente relatado um método validado parcialmente às normas da FDA, em um triplo-quadrupolo LC-MS e um sistema detector HPLC, usando uma coluna C8 (5 µm, identificação de 3,9 mm x 50 mm) com a fase móvel composta de 100% do Grupo ACN e 0,1% ácido fórmico em água (40: 60 v/v) em uma taxa de fluxo de 1 mL/min. Uma correlação linear dos picos observou-se uma faixa de concentração de 0,01 a 10 µ g/mL no plasma humano para todos os metabólitos, ivacaftor, Iva-M1, Iva-M6 e lumacaftor1...

Watch this Scientific Journal Video about Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cys

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Combinação de Ivacaftor e ivacaftor-lumacaftor são duas novas drogas CF. No entanto, ainda há uma falta de entendimento sobre sua PK/PD e farmacologia. Apresentamos uma técnica de HPLC-MS otimizada para a análise simultânea de ivacaftor e seus principais metabolitos e lumacaftor.

Método otimizado de LC-MS/MS para a análise do elevado-throughput de amostras clínicas de Ivacaftor, seus principais metabolitos e Lumacaftor em fluidos biológicos de pacientes de fibrose cística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

optimized-lc-msms-method-for-high-throughput-analysis-clinical

Research

JoVE Journal

Medicine

8.2K visualizações.  Monash University. Combinação de Ivacaftor e ivacaftor-lumacaftor são duas novas drogas CF. No entanto, ainda há uma falta de entendimento sobre sua PK/PD e farmacologia. Apresentamos uma técnica de HPLC-MS otimizada para a análise simultânea de ivacaftor e seus principais metabolitos e lumacaftor.

Vídeo: Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Quantificação do imunossupressor tacrolimus em manchas de sangue seco utilizando LC-MS / MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Medicina

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Análise de Redes de forame oval Eletrodo Recordings na epilepsia do lobo temporal Os pacientes resistentes a drogas

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inchaço na Organóides intestinal induzida por forscolina: An<em&gt; In Vitro</em&gt; Ensaio para a Avaliação de Medicamentos Response na Fibrose Cística Os pacientes

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análise otimizada de<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; A esteatose hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Estratégias de amostragem e processamento de amostras de tecido do Biobanco de suínos modelos biomédicos

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

Coletar amostras de cabelo para cabelo análise de Cortisol em americanos africanos

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Análise de alto throughput de dados de mapeamento óptico usando o ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

Um método de alta produtividade in situ para estimação da ploidiii y nuclear hepatocida em camundongos

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Painel LC-MS/MS validado para quantificar 11 medicamentos resistentes a medicamentos para tuberculose em amostras de cabelos pequenos

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Projeto e Desenvolvimento de um Modelo para Estudar o Efeito do Oxigênio Suplementar no Microbioma da Fibrose Cística

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...