J.L. y T.V. son apoyados por el Instituto Nacional de alergias y enfermedades infecciosas (NIAID) de los institutos nacionales de salud (R01 AI111965). El contenido es responsabilidad exclusiva de los autores y no representan necesariamente la opinión oficial del Instituto Nacional de alergias y enfermedades infecciosas o los institutos nacionales de salud. MC es un investigador australiano NHMRC Principal. J. L. es nacional australiano de salud y Consejo de investigación médica (NHMRC) Senior Research Fellow, y T.V. es un australiano NHMRC industria carrera desarrollo nivel 2 investigador. E.K.S es un 2017 joven Embajador designado para ASM (sociedad estadounidense de Microbiología) y es apoyado por la concesión australiana del postgrado. Partes de este trabajo fue presentado en la 12th Conferencia incesante en la Fibrosis quística en Melbourne (5-8th de agosto de 2017).

aprobación de ética se obtuvo de Monash University humano investigación ética Comité (MUHREC).
 1. aplicación de la prueba: toma de muestras de paciente <ol><li>registrar el tiempo cuando el paciente toma la dosis estándar de 150 mg el ivacaftor o ivacaftor 125 mg /lumacaftor 200 mg.</li>
	<li>Nota abajo el tiempo exacto cuando se recoge la muestra de sangre del paciente. 
	Nota: Se recomienda recoger muestras de 4-5 en un transcurso de tiempo 24 h. Si la colección de solo muestra es posible, el momento indicado es 2.5-4 h post ivacaftor o ivacaftor-lumacaftor combinación de dosificación como C máximo las concentraciones en estado estacionario será alcanzables después de &#62; 5 días de tratamiento consecutivo.</li>
	<li>Recoger 4-5 mL de sangre en tubos azul sin tratamiento disponibles en el mercado.</li>
	<li>Después de la recolección de la sangre, que la sangre coagule dejando tranquilo a temperatura ambiente. 
	Nota: Esto generalmente toma 15-30 minutos</li>
	<li>Eliminar el coágulo por centrifugación a 1.000-2.000 x g durante 10 min a 4 ° C en una centrífuga refrigerada. Designar el sobrenadante resultante como plasma.</li>
	<li>Después de centrifugación, inmediatamente transferir el sobrenadante (plasma) en un tubo limpio de polipropileno con una pipeta Pasteur. Etiqueta el plasma del tubo con el ID del paciente (p. ej., paciente 1), el medicamento administrado (por ejemplo, combinación de ivacaftor-lumacaftor) y tiempo de la muestra del paciente fue recogida (p. ej., dosis después de 2,5 h).</li>
	<li>Mantener las muestras a 2-8 ° C durante la manipulación. Una vez terminado el tratamiento, almacenar las muestras en &#8211; 20 ° C.</li>
	<li>Si es necesario, enviar las muestras con un mensajero aprobado al Instituto análisis. 
	Nota: Las muestras deben enviarse en hielo seco hasta su llegada y debe mantenerse en &#8211; 80° C hasta análisis.</li>
</ol> 2. Preparación y procesamiento de muestras efectuados y normas Nota: Plasma de donantes sanos ingenuos al ivacaftor/lumacaftor tratamiento se obtuvo de la Cruz Roja australiana. Para asegurar la integridad, todas las muestras y normas debe mantenerse a 2-8 ° C durante la recolección y procesamiento. Una vez terminado el tratamiento, almacenar las muestras en &#8211; 20 ° C.
<ol><li>Pesar IVA, IVA-carboxylate, hidroximetil-IVA y LUMA que servirá como referencia para los estándares internos.</li>
	<li>Preparar dos soluciones independientes de cada analito (IVA, M1, M6 y LUMA) en metanol de grado LC-MS en 100 μg/mL y 10 μg/ml. 
	Nota: Los compuestos expirará después de 10 días en el escritorio.</li>
	<li>Recién preparar LC-MS, patrones de calibración antes de cada serie analítica mediante la dilución de los estándares de calibración soluciones en plasma humano para lograr a raíz de las concentraciones: 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 y 10.0 μg/mL. Estas muestras se denominan normas.</li>
	<li>Para precipitar proteínas, preparar 0.1% de ácido fórmico (FA) en acetonitrilo (ACN, grado de LC-MS) y deja en el refrigerador hasta que sea necesario.</li>
</ol> 3. Tratamiento previo de muestras efectuados y normas <ol><li>tanto muestras de plasma del paciente y en blanco se equilibren a temperatura ambiente.</li>
	<li>Vortex para mezclar cada muestra de plasma de 15 s por ejemplo.</li>
	<li>Transfiera una alícuota de 100 μl de cualquiera plasma sin estándares o muestras para análisis en un tubo de microcentrífuga de polipropileno de 1,5 mL.</li>
	<li>Añadir el IVA estándar interno en cada uno de los tubos estándar (por ejemplo, 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 y 10.0 μg/mL para cubrir el potencial rango de concentración), cierre la tapa y vortex por 2-3 s. Nota esa norma interna es sólo Añadida al plasma en blanco y no estándar interno se agrega a las muestras del paciente.</li>
	<li>Repita el paso 3.4. de los otros estándares internos M1, M6 y LUMA.</li>
	<li>Vuelta abajo cada tubo brevemente para asegurar que hay no hay gotas en la tapa de.</li>
	<li>Añadir 200 μL de la mezcla de 0.1% FA en ACN en cada tubo para precipitar las proteínas del plasma.</li>
	<li>Vórtice la mezcla vigorosamente durante 15 s.</li>
	<li>Tubos reposar 10 minutos en el refrigerador.</li>
	<li>Centrifugar a 10.000 x g durante 10 min a 4 ° C, si es posible.</li>
	<li>Filtrar una alícuota de 200 μL del sobrenadante a través de un filtro de la jeringuilla de 13 m m en un frasco HPLC de 1.5 mL.</li>
	<li>Transferir 100 μl del sobrenadante en el frasco de LC-MS para el análisis de HPLC-MS.</li>
</ol> 4. El análisis por HPLC-MS Nota: HPLC-MS el análisis fue realizado en un sistema LC-MS juntado con el espectrómetro de masas de triple cuadrupolo (tabla 1).
<ol><li>Vuelta en el sistema LC-MS, coloque la bandeja con las muestras en el dechado auto.</li>
	<li>Fijar la columna en una columna de guardia y conectar al sistema LC-MS.</li>
	<li>Conectar dos botellas de fases móviles (botella A: 100% ACN; botella B: 0,1% ácido fórmico en agua) y equilibrar el sistema LC-MS.</li>
	<li>Incorporar a los siguientes parámetros en el protocolo de LC-MS.
	<ol><li>Divide el flujo de fase móvil antes de entrar en el espectrómetro de masas en una proporción 2:1 (residuos: entrada de MS).</li>
		<li>Realizar una elución gradiente a un flujo de 0,5 mL/min utilizando una fase móvil que consiste en 100% ACN y 0.1% de ácido fórmico en agua (40: 60, el punto de partida v/v). Tenga en cuenta que el porcentaje de volumen de la fase móvil cambia a lo largo del análisis.</li>
		<li>Operar el espectrómetro de masas en modo de ionización por electrospray positivo.</li>
		<li>Asegúrese de que los valores de LC-MS son los siguientes: voltaje de spray ion 4,5 kV, energía de la colisión 295.9 V, nebulización gas: nitrógeno a 3 L/min, gas de colisión: argón; secado gas caudal 20 L/min, voltaje Q3 de la lente:-22 V, desolvation temperatura 250 ° C con un bloque de calor temperatura de 400 ° C.</li>
		<li>Volumen de inyectar 10 μl por muestra.</li>
		<li>Detectar analitos usando la reacción múltiple (MRM) de monitoreo. Seguimiento de las transiciones del ion de m/z 392.49 &#8594; 393, m/z 408.49 &#8594; 409, m/z 422.47 &#8594; 423 y m/z 452.40 &#8594; 453 para IVA, IVA-M1, M6 IVA y LUMA, respectivamente. 
		Nota: El método recientemente optimizado aún tiene que ser plenamente validado según estándares de la FDA.</li>
	</ol></li></ol> 5. Curvas de calibración <ol><li>construir la LC-MS curvas de calibración antes de cada serie analítica usando la relación entre las proporciones de área de pico de cada uno de los cuatro analitos a estándar interno y las calibración estándar nominal las concentraciones de El ivacaftor (IVA-M1, M6 IVA o LUMA) dentro del programa de configuración de LC-MS. 
	Nota: Los pasos exactos para la construcción de la curva de calibración es dependiente en el modelo de equipo LC-MS se utiliza. Esta información puede estar disponible en el manual del equipo.</li>
	<li>Realizar el análisis de regresión lineal de mínimos cuadrados ponderando 2 según el recíproco de las concentraciones de.</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>

Los autores no tienen nada que revelar.

Como se informó anteriormente, nuestro grupo tiene por primera vez desarrollado y validado un método de HPLC y LC-MS para la detección y cuantificación de ivacaftor y sus principales metabolitos hidroximetil-IVA M1 (activo) y el IVA-carboxylate M6 (inactivos); y lumacaftor en el plasma y el esputo de pacientes de CF14. El análisis divulgado previamente por nuestro grupo fue utilizado con éxito para cuantificar la concentración de LUMA, IVA, IVA-M1 y M6 de IVA en el plasma y el esputo de pac...

La fibrosis quística (FQ) es una enfermedad genética común que implica las glándulas exocrinas de la mucosa del pulmón, hígado, páncreas y los intestinos causando insuficiencia multiorgánica progresiva, como una disminución en la función pulmonar y la insuficiencia pancreática1, 2,3. Ivacaftor (IVA) es el primer Food and Drug Administration (FDA-US) y Agencia Europea de medicamentos (EMA) aprobó fibrosis quística trans membrana conductancia regulador (CFTR) potenciador de drogas, con eficacia clínica se manifiesta produciendo un significativo mejora la función pulmonar en comparación con placebo en un pequeño subgrupo de pacientes con FQ teniendo la G551D-CFTR [glicina (G) en posición 551 es reemplazado por ácido aspártico (D)] mutación sin sentido (~ 4-5% de la población de CF)4,5. Esto administrados por vía oral drogas aumenta el canal CFTR abriendo, así aumentar el flujo de iones de cloruro y actuando sobre el defecto primario que conduce a las manifestaciones clínicas de CF4,6. Lamentablemente, IVA monoterapia no es eficaz en pacientes con la mutación más común de F508del homocigótica [en canceladura del marco del gen CFTR que se traduce en la pérdida de la fenilalanina (F) en la posición 508] que resulta en mal plegada CFTR, que se observa en ~ 50% de la población de CF7,8.
Recientemente, la FDA otorgó la aprobación para combinar IVA con el corrector CFTR lumacaftor drogas. La inteligente estrategia de combinar un corrector CFTR (lumacaftor LUMA) que rescata la F508del-CFTR a la superficie de la célula con un modulador (IVA) que potencia la actividad del canal CFTR, efectivamente se expande la ventana de tratamiento a la mayoría de la población de CF5 . Sigue habiendo preguntas sobre si estos fármacos cumplirá su promesa como un número de informes contradictorios han emergido poner en duda sobre su eficacia clínica9,10. Además, mejoras en la función pulmonar fueron sólo modesto (2.6-4% para combinación ivacaftor-lumacaftor) en comparación con el éxito alcanzado con la monoterapia en pacientes con una mutación (10.6-12,5%) de G551D8de IVA. Posibles interacciones de fármacos antagonistas entre IVA y LUMA que limitan potencialmente la eficacia clínica de la combinación lumacaftor ivacaftor provienen de sus propiedades farmacocinéticas ideal menos de7,11. IVA es ampliamente metabolizada por las enzimas del citocromo P450 (CYP), principalmente a un metabolito activo hidroximetil-IVA (IVA-M1, M1) y una forma inactiva de7,IVA-carboxylate (IVA-M6, M6)12. El inductor de CYP3A4 LUMA, por otro lado, no se metaboliza extensamente y en gran parte se excreta inalterado en la heces11. Como inductores de CYP3A4 inducen metabolismo del citocromo, se podrían reducir concentraciones ivacaftor (sustrato de CYP3A4). Por otra parte, tanto el IVA como LUMA son moléculas muy hidrofóbicas y están obligado ~ 99% a proteínas plasmáticas, que limita significativamente la concentración de libre de drogas (activo)1,13.
Colectivamente, estos factores pueden ser que se reúnen para limitar la eficacia clínica de la combinación de ivacaftor-lumacaftor. No se sabe si se alcanzan las concentraciones plasmáticas óptimas bajo el actual régimen de dosificación para el ivacaftor-lumacaftor combinación o si el umbral terapéutico se mantiene8. Actualmente, hay una escasez de información sobre los parámetros farmacocinéticos como el pico y las concentraciones plasmáticas de estado estacionario de ivacaftor o ivacaftor-lumacaftor. Dado el notable metabolismo de ivacaftor y lumacaftor, seguimiento de las relaciones exposición-respuesta es requisito para alcanzar regímenes de dosis óptima para tratamiento ivacaftor o ivacaftor-lumacaftor. Nuestro grupo ha publicado recientemente el primer método HPLC, LC-MS para la supervisión de las relaciones exposición-respuesta de IVA y LUMA14. No hay técnicas alternativas de medición de las concentraciones de ivacaftor, sus metabolitos y lumacaftor se han divulgado hasta la fecha. Para permitir el análisis de alto rendimiento de un colectivo de pacientes mayor y para reducir drásticamente el tiempo de análisis, nuestro grupo ha optimizado el método divulgado a través de una columna de cromatografía de fase inversa de tamaño de poro más pequeño y un sistema solvente degradado que reduce costos y tiempos en marcha.

<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

Defectos en el regulador de conductancia trans membrana de fibrosis quística (CFTR) son la causa de la fibrosis quística (FQ), una enfermedad con manifestaciones pulmonares mortales. El ivacaftor (IVA) y combinación de ivacaftor-lumacaftor (LUMA) son dos nuevos medicamentos avance CF que modulan directamente la actividad y el tráfico de la proteína CFTR defectuosa. Sin embargo, todavía hay una falta de entendimiento en los parámetros farmacocinéticos/farmacodinámicos y la farmacología de ivacaftor y lumacaftor. La técnica de HPLC-MS para el análisis simultáneo de las concentraciones de ivacaftor, hidroximetil-ivacaftor, ivacaftor-carboxylate y lumacaftor en fluidos biológicos en pacientes que reciben la combinación estándar de ivacaftor o ivacaftor-lumacaftor la terapia ha sido desarrollada por nuestro grupo y validado parcialmente a las normas de la FDA. Sin embargo, para permitir el análisis de alto rendimiento de un mayor número de muestras de pacientes, nuestro grupo ha optimizado el método divulgado a través de una columna de cromatografía de fase inversa de tamaño de poro más pequeño (2.6 μm, C8 100 Å; 50 x 2,1 mm) y un sistema solvente degradado ( 0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: en 70% B; 2.7-2,8 min: 70-90% de B; 2,8-4,0 min: 90% lavado de B; 4.0-4,1 min: 90-40% B; 4.1 6.0 min: en 40% B) en lugar de una elución isocrática. El objetivo de este estudio era reducir el tiempo de análisis HPLC-MS por muestra dramáticamente de ~ 15 minutos a sólo 6 minutos por muestra, que es esencial para el análisis de una gran cantidad de muestras de pacientes. Este método conviene será de gran utilidad para estudios en las relaciones exposición-respuesta de estas drogas de CF de avance.

Recientemente hemos reportado un método, validado parcialmente a las normas de la FDA, en un triple cuadrupolo LC-MS y un sistema de detector HPLC, usando una columna C8 (5 μm, 3,9 x 50 mm diámetro interior) con la fase móvil que consiste en 100% ACN y 0.1% de ácido fórmico en agua (40: 60 v/v) con un caudal de 1 mL/min. Se observó una correlación linear de los picos sobre un rango de concentración de 0,01 a 10 μg/mL en plasma humano para todos los metabolitos, ivacaftor, Iva-M1, M6 Iva y lumacaftor<sup class="...

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

Combinación de ivacaftor y ivacaftor lumacaftor son dos nuevos fármacos de CF. Sin embargo, todavía hay una carencia de entendimiento sobre su PK/PD y farmacología. Presentamos una técnica de HPLC-MS optimizada para el análisis simultáneo de ivacaftor y sus principales metabolitos y lumacaftor.

Método optimizado de LC-MS/MS para el análisis de alto rendimiento de las muestras clínicas de Ivacaftor, sus principales metabolitos y Lumacaftor en fluidos biológicos de pacientes con Fibrosis quística

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 vistas.  Monash University. Combinación de ivacaftor y ivacaftor lumacaftor son dos nuevos fármacos de CF. Sin embargo, todavía hay una carencia de entendimiento sobre su PK/PD y farmacología. Presentamos una técnica de HPLC-MS optimizada para el análisis simultáneo de ivacaftor y sus principales metabolitos y lumacaftor.

Video: 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.

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

Investigación

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.

Análisis de la red de foramen oval de electrodos Grabaciones en epilepsia del lóbulo temporal pacientes resistentes a los medicamentos

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.

Inducida por la forskolina Hinchazón en organoides intestinal: Una<em&gt; In Vitro</em&gt; Ensayo para evaluar la respuesta de drogas en pacientes con fibrosis quística

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.

Análisis optimizada de<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; La esteatosis 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.

Estrategias de muestreo y procesamiento de las muestras de tejido de Biobanco de porcinos 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.

Recoger muestras de pelo para cabello análisis de Cortisol en 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.

Análisis de alto rendimiento de datos de asignación óptica mediante 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.

Un método in situ de alto rendimiento para la estimación de la ploideide nuclear de hepatocitos en ratones

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.

Panel LC-MS/MS validado para cuantificar 11 medicamentos resistentes a la tuberculosis en muestras de pelo pequeño

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.

Diseño y desarrollo de un modelo para estudiar el efecto del oxígeno suplementario en el microbioma de las vías respiratorias de la fibrosis quística

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

Método optimizado de LC-MS/MS para el análisis de alto rendimiento de las muestras clínicas de Ivacaftor, sus principales metabolitos y Lumacaftor en fluidos biológicos de pacientes con Fibrosis quística

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