Name: 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 (Video) | JoVE Journal
Uploaded: 2017-10-15T13:00:00.000+00:00
Duration: 6 min 14 s
Description: Learn an optimized LC-MS/MS method for high-throughput analysis of ivacaftor, its metabolites, and lumacaftor in cystic fibrosis patient plasma. This sensitive approach enables reliable therapeutic drug monitoring and pharmacokinetic studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

J.L. and T.V. are supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (R01 AI111965). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. MC is an Australian NHMRC Principal Research Fellow. J. L. is an Australian National Health and Medical Research Council (NHMRC) Senior Research Fellow, and T.V. is an Australian NHMRC Industry Career Development Level 2 Research Fellow. E.K.S is an appointed Young Ambassador 2017 for ASM (American Society for Microbiology) and is supported by the Australian Postgraduate Award. 
Parts of this work was presented at the 12th Australiasian Conference on Cystic Fibrosis In Melbourne (5-8th of August 2017).

Approval for ethics was obtained from Monash University Human Research Ethics Committee (MUHREC).
1. Application of the Assay: Patient Sample Collection
<ol>
	<li>Record the time when the patient takes their standard dose of either 150 mg ivacaftor or ivacaftor 125 mg /lumacaftor 200 mg.</li>
	<li>Note down the exact time when the patient blood sample is collected. 
	NOTE: We recommend collecting 4-5 samples over a 24 h time course. If the collection of only one sample is possible, the indicated time point is 2.5-4 h post ivacaftor or ivacaftor-lumacaftor combination dosing as Cmax concentrations at steady state will be achievable after &#62;5 days of consecutive treatment.</li>
	<li>Collect 4-5 mL of blood in commercially available untreated blue tubes.</li>
	<li>After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. 
	NOTE: This usually takes 15-30 min.</li>
	<li>Remove the clot by centrifuging at 1,000-2,000 x g for 10 min at 4 &#176;C in a refrigerated centrifuge. Designate the resulting supernatant as plasma.</li>
	<li>Following centrifugation, immediately transfer the supernatant (plasma) into a clean polypropylene tube using a Pasteur pipette. Label the plasma tube with the Patient ID (e.g., Patient 1), the administered drug (e.g., ivacaftor-lumacaftor combination), and time the patient sample was collected (e.g., 2.5 h post-dosage).</li>
	<li>Maintain samples at 2-8 &#176;C during handling. Once handling is completed, store samples at &#8211;20 &#176;C.</li>
	<li>If necessary, ship samples with an approved courier to the analyzing institute. 
	NOTE: Samples are to be shipped on dry ice until arrival and should be kept at &#8211;80&#176;C until analysis.</li>
</ol>
2. Preparation and Processing of Incurred Samples and Standards
NOTE: Plasma from healthy donors na&#239;ve to ivacaftor/lumacaftor therapy was obtained from the Australian Red Cross. To ensure the integrity, all samples/standards should be kept at 2-8 &#176;C during collection and processing. Once handling is completed, store samples at &#8211;20 &#176;C.
<ol>
	<li>Weigh out IVA, IVA-carboxylate, hydroxymethyl-IVA, and LUMA which will serve as a reference for the internal standards.</li>
	<li>Prepare two independent stock solutions of each analyte (IVA, M1, M6 and LUMA) in LC-MS grade methanol at 100 &#181;g/mL and at 10 &#181;g/mL. 
	Note: Compounds will expire after 10 days on the benchtop.</li>
	<li>Freshly prepare LC-MS calibration standards prior to each analytical run by dilution of the calibration standards stock solutions into human plasma to achieve following concentrations: 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, and 10.0 &#181;g/mL. These samples are referred to as standards.</li>
	<li>To precipitate proteins, prepare 0.1% formic acid (FA) in acetonitrile (ACN, LC-MS grade) and leave in the refrigerator until needed.</li>
</ol>
3. Pre-treatment of Incurred Samples and Standards
<ol>
	<li>Allow both patient and blank plasma samples to equilibrate to room temperature.</li>
	<li>Vortex to mix each plasma sample for 15 s per sample.</li>
	<li>Transfer a 100 &#181;L aliquot of either blank plasma for standards or patient samples for analysis into a 1.5-mL polypropylene microcentrifuge tube.</li>
	<li>Add the internal standard IVA into each of the standard tubes (e.g., 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, and 10.0 &#181;g/mL to cover the potential concentration range), close the lid, and vortex for 2-3 s. Note that internal standard is only added to the blank plasma and no internal standard is added to the patient samples.</li>
	<li>Repeat step 3.4. for the other internal standards M1, M6, and LUMA.</li>
	<li>Spin down each tube briefly to ensure that there are no droplets on the lid.</li>
	<li>Add 200 &#181;L of the mix of 0.1% FA in ACN into each tube to precipitate plasma proteins.</li>
	<li>Vortex the mixture vigorously for about 15 s.</li>
	<li>Allow tubes to stand for 10 min in the refrigerator.</li>
	<li>Centrifuge at 10,000 x g for 10 min at 4 &#176;C, if possible.</li>
	<li>Filter a 200 &#181;L aliquot of the supernatant through a 13 mm syringe filter into a 1.5 mL HPLC vial.</li>
	<li>Transfer 100 &#181;L of the supernatant into the LC-MS vial for the HPLC-MS analysis.</li>
</ol>
4. HPLC-MS Analysis
NOTE: The HPLC-MS analysis was performed on an LC-MS system coupled with the triple quadrupole mass spectrometer (Table 1).
<ol>
	<li>Turn on the LC-MS system, place the tray with the samples in the auto-sampler.</li>
	<li>Attach the column to a guard column and connect to the LC-MS system.</li>
	<li>Attach both bottles of mobile phases (bottle A: 100% ACN; bottle B: 0.1% formic acid in water) and equilibrate the LC-MS system.</li>
	<li>Incorporate the following parameters into the LC-MS protocol.
	<ol>
		<li>Split the mobile phase flow before entering the mass spectrometer in a 2:1 ratio (waste: MS inlet).</li>
		<li>Perform a gradient elution at a flow rate of 0.5 mL/min using a mobile phase consisting of 100% ACN and 0.1% formic acid in water (starting point at 40:60, v/v). Note that the volume percent of the mobile phase changes throughout analysis.</li>
		<li>Operate the mass spectrometer in a positive electrospray ionization mode.</li>
		<li>Make sure that the LC-MS settings are as follows: ion spray voltage 4.5 kV, collision energy 295.9 V, nebulizing gas: nitrogen at 3 L/min; collision gas: argon; drying gas flow 20 L/min, lens voltage Q3: - 22 V, desolvation temperature 250 &#176;C with a heat block temperature of 400 &#176;C.</li>
		<li>Inject 10 &#181;L volume per sample.</li>
		<li>Detect analytes using multiple reaction monitoring (MRM). Monitor the ion transitions of m/z 392.49 &#8594; 393, m/z 408.49 &#8594; 409, m/z 422.47 &#8594; 423 and m/z 452.40 &#8594; 453 for IVA, IVA-M1, IVA-M6 and LUMA, respectively. 
		NOTE: The newly optimized method has yet to be fully validated according to FDA standards.</li>
	</ol>
	</li>
</ol>
5. Calibration Curves
<ol>
	<li>Construct the LC-MS calibration curves before each analytical run using the relationship between the peak area ratios of each of the four analytes to internal standard and the calibration standard nominal concentrations of ivacaftor (IVA-M1, IVA-M6 or LUMA) within the LC-MS settings program. 
	NOTE: The exact steps for the construction of the calibration curve is dependent on the model of LC-MS equipment being used. This information may be available in the equipment manual.</li>
	<li>Perform linear least-squares regression analysis by weighting 1/C2 according to the reciprocal of concentrations.</li>
</ol>

<ol><li>Schneider, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug-drug+plasma+protein+binding+interactions+of+ivacaftor%5BTitle%5D)+AND+%22J+Mol+Recognit%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSolomon%2C+M+%22Treatments+for+Cystic+fibrosis%22">Treatments for Cystic fibrosis</a>. , (2009).</li>
<li>O'Sullivan, B. P., Flume, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+clinical+approach+to+lung+disease+in+patients+with+cystic+fibrosis%5BTitle%5D)+AND+%22Semin+Respir+Crit+Care+Med%22%5BJournal%5D)">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/pubmed?term=((A+CFTR+potentiator+in+patients+with+cystic+fibrosis+and+the+G551D+mutation%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">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/pubmed?term=((Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">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/pubmed?term=((Discovery+of+N-%282%2C4-di-tert-butyl-5-hydroxyphenyl%29-4-oxo-1%2C4-dihydroquinoline-3-carboxamide+%28VX-770%2C+ivacaftor%29%2C+a+potent+and+orally+bioavailable+CFTR+potentiator%5BTitle%5D)+AND+%22J+Med+Chem%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22FDA+Advisory+Commitee+Briefing+Material+VERTEX-FDA+Pulmonary-Allergy+drugs+advisory+commitee%22">FDA Advisory Commitee Briefing Material VERTEX-FDA Pulmonary-Allergy drugs advisory commitee</a>. 98 , VERTEX Pharmaceuticals Incorporated. (2015).</li>
<li>Holmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((False+dawn+for+cystic+fibrosis+disease+modifiers%3F%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">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/pubmed?term=((Some+gating+potentiators%2C+including+VX-770%2C+diminish+DeltaF508-CFTR+functional+expression%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">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/pubmed?term=((Potentiator+ivacaftor+abrogates+pharmacological+correction+of+DeltaF508+CFTR+in+cystic+fibrosis%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">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.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/003954/human_med_001935.jsp&mid=WC0b01ac058001d124">Assessment report ORKAMBI (ivacaftor/lumacaftor) European medicines agency</a>. , (2015).</li>
<li>VERTEX. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Vertex+prescribing+infomation%22">Vertex prescribing infomation</a>. , (2015).</li>
<li>Matthes, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+free+drug+concentration+prevents+inhibition+of+F508del+CFTR+functional+expression+by+the+potentiator+VX-770+%28ivacaftor%29%5BTitle%5D)+AND+%22Br+J+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Development+of+HPLC+and+LC-MS%2FMS+methods+for+the+analysis+of+ivacaftor%2C+its+major+metabolites+and+lumacaftor+in+plasma+and+sputum+of+cystic+fibrosis+patients+treated+with+ORKAMBI+or+KALYDECO%5BTitle%5D)+AND+%22J+Chrom+B+Analyt+Technol+Biomed+Life+Sci%22%5BJournal%5D)">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/pubmed?term=((An+LC-MS%2FMS+method+for+the+quantitation+of+cabozantinib+in+rat+plasma%3A+application+to+a+pharmacokinetic+study%5BTitle%5D)+AND+%22J+Chromatogr+B+Analyt+Technol+Biomed+Life+Sci%22%5BJournal%5D)">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>

The authors have nothing to disclose.

As previously reported, our group has for the first time developed and validated a HPLC and LC-MS method for rapid detection and quantification of ivacaftor and its major metabolites hydroxymethyl-IVA M1 (active) and IVA-carboxylate M6 (inactive); and lumacaftor in the plasma and sputum of CF patients14. The assay reported by our group previously was successfully used to quantify the concentration of LUMA, IVA, IVA-M1, and IVA-M6 in the plasma and sputum of CF patients undergoing steady state stan...

Cystic fibrosis (CF) is a common genetic disease involving the exocrine mucus glands of the lung, liver, pancreas, and intestines causing progressive multi-organ failure, such as a decline in lung function and pancreatic insufficiency1,2,3. Ivacaftor (IVA) is the first Food and Drug Administration (FDA-US) and European Medicines Agency (EMA) approved cystic fibrosis trans-membrane conductance regulator (CFTR) potentiator drug, with evidenced clinical efficacy producing a significant improvement in the lung function over placebo in a small subset of CF patients bearing the G551D-CFTR [glycine (G) in position 551 is replaced by aspartic acid (D)]&#160;missense mutation (~4-5% of the CF population)4,5. This orally administered drug increases the CFTR channel opening, thus increasing the chloride ion flow and acting on the primary defect that leads to the clinical manifestations of CF4,6. Unfortunately, IVA monotherapy is not effective in patients with the more common homozygous F508del mutation [in frame deletion of the CFTR gene which results in the loss of phenylalanine (F) at position 508]&#160;which results in misfolded CFTR, which is seen in ~50% of the CF population7,8.
Recently, the FDA has granted approval for combining IVA with the CFTR corrector drug lumacaftor. The clever strategy of combining a CFTR corrector (lumacaftor, LUMA) which rescues F508del-CFTR to the cell surface with a modulator (IVA) which potentiates CFTR channel activity, effectively expands the treatment window to most of the CF population5. Questions remain over whether these drugs will fulfill their promise as a number of conflicting reports have emerged that cast doubt upon their clinical efficacy9,10. Additionally, improvements in lung function were only modest (2.6-4% for ivacaftor-lumacaftor combination) compared to the success achieved with IVA monotherapy in patients bearing a G551D mutation (10.6-12.5%)8. Potential antagonistic drug-drug interactions between IVA and LUMA that potentially limit the clinical efficacy of ivacaftor-lumacaftor combination come from its less than ideal pharmacokinetic properties7,11. IVA is extensively metabolized by cytochrome P450 enzymes (CYP), primarily to an active metabolite hydroxymethyl-IVA (IVA-M1, M1) and an inactive form IVA-carboxylate (IVA-M6, M6)7,12. The CYP3A4 inducer LUMA, on the other hand, is not extensively metabolized and is largely excreted unchanged in the feces11. As CYP3A4 inducers induce cytochrome metabolism, ivacaftor (CYP3A4 substrate) concentrations could be reduced. Moreover, both IVA and LUMA are very hydrophobic molecules and are ~99% bound to plasma proteins, which significantly limits the free (active) drug concentration1,13.
Collectively, these factors may be coming together to limit the clinical efficacy of ivacaftor-lumacaftor combination. It is not known whether optimal plasma concentrations are achieved under the current dosage regimen for ivacaftor-lumacaftor combination or if the therapeutic threshold is maintained8. Presently, there is a paucity of information regarding pharmacokinetic parameters such as the peak and steady-state plasma concentrations of ivacaftor or ivacaftor-lumacaftor. Given the noted metabolism of ivacaftor and lumacaftor, monitoring of exposure-response relationships is requisite to achieve optimal dosage regimens for ivacaftor or ivacaftor-lumacaftor therapy. Our group recently published the first HPLC/LC-MS method for the monitoring of exposure-response relationships of IVA and LUMA14. No alternative techniques of measuring the concentrations of ivacaftor, its metabolites, and lumacaftor have been reported to date. To allow the high-throughput analysis of a larger patient collective and to dramatically reduce analysis time, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column and a gradient solvent system that reduces cost and running times.

<table><tbody><tr><td>IVA (Cat#S114)&amp;nbsp;</td><td>SelleckChem (USA).&amp;nbsp;</td><td></td><td></td></tr><tr><td>LUMA (Cat#S1565)&amp;nbsp;</td><td>SelleckChem (USA).&amp;nbsp;</td><td></td><td></td></tr><tr><td>IVA-carboxylate (Cat# 510242247CS)&amp;nbsp;</td><td>Clearsynth (Canada).&amp;nbsp;</td><td></td><td></td></tr><tr><td>hydroxymethyl-IVA (Cat# 510240849CS)&amp;nbsp;</td><td>Clearsynth (Canada).&amp;nbsp;</td><td></td><td></td></tr><tr><td>Methanol (MeOH, LC-MS grade),&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>acetonitrile (ACN, LC-MS grade)&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>formic acid (FA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>triple-quadrupole Shimadzu 8030 LC-MS&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Phenomenex Kinetex (2.6 &amp;micro;m C8 100 &amp;Aring;; 50 &amp;times; 2.1mm)</td><td></td><td></td><td></td></tr><tr><td>(KrudKatcher Ultrea HPLC In-Line Filter 0.5 m Depth Filter x 0.004inID).&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>1.5 mL polypropylene microcentrifuge tube (VWR).&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Eppendorf Centrifuge 5430</td><td></td><td></td><td></td></tr><tr><td>13-mm syringe filter (0.45 &amp;micro;m nylon, GRACE, USA)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>[Phenomenex VEREX, 9 mm, PP, 300 &amp;micro;L, PTFE/Silicone septa].&amp;nbsp;</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

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

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

We have recently reported a method, partially validated to FDA standards, on a triple-quadrupole LC-MS and an HPLC detector system, using a C8 column (5 µm, 3.9 mm x 50 mm i.d.) with the mobile phase consisting of 100% ACN and 0.1% formic acid in water (40:60, v/v) at a flow rate of 1 mL/min. A linear correlation of the peaks was observed over a concentration range from 0.01 to 10 µg/mL in human plasma for all metabolites, ivacaftor, Iva-M1, Iva-M6, and lumacaftor14. Here this method has been opti...

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

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

This is the protocol for the sample preparation for the plasma assay. Please note, to ensure their integrity, all samples and standards should be kept at two to eight degrees Celsius during collection and processing. Once handling is completed, store all samples at minus 20 degrees.For the standard preparation, prepare two independent stock solution of each anilide, ivacaftor, M1 metabolite, M6 metabolite, and lumacaftor in methanol at 100 microgram per milliliter and 10 microgram per milliliter. Please note, the compounds expire after 10 days days on bench top. For the standards, transfer 100 microliters of human plasma into the 1.5 milliliter polypropylene microcentrifuge tubes.Continue to do so for all your other samples. Then add the internal standard to each tube. For example, we choose 0.01 microgram per milliliters out of the prepared standard.Now this tube contains plasma spiked with the internal standard. Proceed like this with all other standards. Take your standards and spin it down for a few seconds to make sure that all liquid is at the bottom of the tube.Continue to do this for all your 40 standards. For the patient samples, transfer 100 microliters out of the patient sample tube into the microcentrifuge tube. The real patient sample is not spiked with internal standard.Spin the patient sample down for a few seconds to make sure that all liquid is at the bottom of the tube. The next steps are both designed for the samples and the standards. Add 200 microliters of 0.1%formic acid in acetonitrile into each sample tube to precipitate plasma proteins.Continue to do so for all standards and patient samples. Vortex each sample vigorously for about 15 seconds. Continue to do so for all standards and incurred samples.Allow to stand for 10 minutes in the fridge to facilitate precipitation. For demonstrating purposes, I will only centrifuge one sample and one standard. We are centrifuging at 10, 000 rpm for 10 minutes on an Eppendorf Centrifuge 5430 at four degrees.We will now filter the supernatant into the Phenomenex vials. For this, we are using a 13 millimeter syringe filter with 0.45 micrometers nylon Grace filters purchased from USA, and we're filtering into the HPLC vials that can contain 1.5 milliliters. After filtration, we will transfer 100 microliters of supernatant into the LC/MS vials for LC/MS analysis.This sample is now ready for analysis. Now simply put all your samples into the vial rack. We are using a Shimadzu 8030 LC/MS system, coupled with a triple-quadrupole mass spec.Let the system equilibrate before analysis. Once equilibrated, which takes about 15 minutes, just press the Start button. For each metabolite, construct a calibration curve by plotting the peak area ratios of anilides versus the nominal concentration of calibration standards.Use the regression equation for the calibration curve to calculate the concentrations of anilides in the incurred sample. A typical chromatogram is shown in this figure. Optimized chromatographic resolution was obtained using a mobile phase of 100%acetonitrile and 0.1%formic acid in water with a gradient elution on a Phenomenex Kinetex C8 column.The LC retention times were as follows. For M6, one minute. For M1, 1.3 minutes.For ivacaftor, 1.55 minutes. For lumacaftor, 2.3 minutes. In all blank plasma samples, no interference with the retention time of either of the anilides was observed, nor was interference detected from the combination of ivacaftor with lumacaftor.Through the high-throughput analysis of a large amount of patient samples, we have optimized our reported method through the use of a smaller pore size reversed-phase chromatography column Phenomenex C8 column and a gradient solvent system instead of the current isocratic elution. This reduces the running time to six minutes per sample, including the washing phase. This reliable and novel method offers a simple, sensitive, and rapid approach for the therapeutic drug monitoring of ivacaftor and ivacaftor-lumacaftor combination and biological fluids.Given the need to develop exposure-response relationships to maximize drug efficacy as well as contain healthcare costs, more sensitive tools need to be developed and validated to assist clinicians in using evidence-based therapy and high-throughput analytical techniques for patients suffering from CF.By applying the proposed analytical method to clinical pharmacokinetic and pharmacodynamic studies, the opportunity arises to further investigate the pharmacokinetic parameters of ivacaftor or ivacaftor-lumacaftor combination on a larger patient collective.

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

Research

JoVE Journal

Medicine

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism.  This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

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.

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

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Current methods of analyzing patients’ 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.

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

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Learn an optimized LC-MS/MS method for high-throughput analysis of ivacaftor, its metabolites, and lumacaftor in cystic fibrosis patient plasma. This sensitive approach enables reliable therapeutic drug monitoring and pharmacokinetic studies.

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 (Video) | JoVE Journal

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

Explore More Videos