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Overview In this fast analysis, a direct, in-plate protein precipitation and filtration is applied to increase the throughput of LC-MS/MS measurement for chloroquine, hydroxychloroquine and the metabolite desethylchloroquine from serum. The automatable sample extraction procedure produced excellent recovery values. Sample Preparation Rapid Sample Extraction of Chloroquine, Hydroxychloroquine, and Desethylchloroquine from Serum for LC-MS/MS Analysis Dawn Chen, Shahana Huq, and Laura Snow Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA Page 1 of 2 Column: Dimension: Part No.: Mobile Phase: Gradient: Flow Rate: Temperature: Detection: Sample: Kinetex® 2.6 µm F5 50 x 2.1 mm 00B-4723-AN A: 0.1 % Formic Acid in Water B: Methanol Time (min) %B 0.0 2 2.0 7 3.0 15 3.2 40 4.2 95 6.0 2 0.5 mL/min 20 °C MS/MS (MRM, SCIEX® 4000 QTRAP®) 1. Desethylchloroquine 2. Hydroxchloroquine 3. Chloroquine LC-MS/MS Conditions Have questions or want more details on implementing this method? We would love to help! Visit www.phenomenex.com/ChatNow to get in touch with one of our Technical Specialists 3 1 2 Advantages of the Workflow 1. High-throughput a. 96-well format allows for rapid, 10 minutes sample preparation of 96 samples at once b. Analyze samples by LC-MS/MS in 6 minutes 2. Easily automatable sample preparation a. Effectively hold the organic solvent prior to and during the protein precipitation for easy sample cleanup b. Remove precipitated proteins while sample and Chloroquine and its metabolites pass through and into a collection plate, no transfer steps required Recovery Value (%) and CVs (%) CV (n=6) Recovery 10.6 97.1 Chloroquine (ClQ) 16.9 95.8 Hydroxychloroquine (HClQ) 16.2 103.3 Desethylchloroquine (DesClQ) 96-Well Plate: Impact™ Protein Precipitation Plate Part No.: CE0-7565 Dispense: 450 µL of Methanol into each well Add: 150 µL serum into each well Vortex: 2 minutes Filter: Place an empty collection plate below the Impact protein precipitation plate and pass sample through by applying vacuum, positive pressure, or centrifuge Dilute: Dilute filtrate 1:5 with aqueous 0.1 % Formic acid Inject: Inject onto LC-MS/MS App ID 25942 Revision: PHEN-RUO-00120 0
Transcript of Z ] ^ u o Æ ] } v } ( Z o } } µ ] v U , Ç } Æ Ç Z o } } µ ] v U v Z Ç o Z o ... ·...
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OverviewIn this fast analysis, a direct, in-plate protein precipitation and filtration is applied to increase the throughput of LC-MS/MS measurement for chloroquine, hydroxychloroquine and the metabolite desethylchloroquine from serum. The automatable sample extraction procedure produced excellent recovery values.
Sample Preparation
Rapid Sample Extraction of Chloroquine, Hydroxychloroquine, and Desethylchloroquine from Serum for LC-MS/MS AnalysisDawn Chen, Shahana Huq, and Laura SnowPhenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
Page 1 of 2
Column:Dimension:
Part No.:Mobile Phase:
Gradient:
Flow Rate:Temperature:
Detection:Sample:
Kinetex® 2.6 µm F550 x 2.1 mm00B-4723-ANA: 0.1 % Formic Acid in WaterB: MethanolTime (min) %B0.0 22.0 73.0 153.2 404.2 956.0 20.5 mL/min20 °CMS/MS (MRM, SCIEX® 4000 QTRAP®)1. Desethylchloroquine2. Hydroxchloroquine3. Chloroquine
LC-MS/MS Conditions
Have questions or want more details on implementing this method? We would love to help!Visit www.phenomenex.com/ChatNow to get in touch with one of our Technical Specialists
31
2
Advantages of the Workflow1. High-throughput
a. 96-well format allows for rapid, 10 minutes sample preparation of 96 samples at once
b. Analyze samples by LC-MS/MS in 6 minutes
2. Easily automatable sample preparationa. Effectively hold the organic solvent
prior to and during the protein precipitation for easy sample cleanup
b. Remove precipitated proteins while sample and Chloroquine and its metabolites pass through and into a collection plate, no transfer steps required
Recovery Value (%) and CVs (%)CV (n=6)Recovery
10.697.1Chloroquine (ClQ)16.995.8Hydroxychloroquine (HClQ)16.2103.3Desethylchloroquine (DesClQ)
96-Well Plate: Impact™ Protein Precipitation PlatePart No.: CE0-7565Dispense: 450 µL of Methanol into each wellAdd: 150 µL serum into each wellVortex: 2 minutes
Filter:
Place an empty collection plate below the Impact protein precipitation plate and pass sample through by applying vacuum, positive pressure, or centrifuge
Dilute:Dilute filtrate 1:5 with aqueous 0.1 % Formic acid
Inject: Inject onto LC-MS/MS
App
ID 2
5942
Rev
isio
n:
PH
EN
-RU
O-0
0120
0
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www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country/region, contact Phenomenex USA, International Department at [email protected]
Terms and ConditionsSubject to Phenomenex Standard Terms and Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions.TrademarksKinetex is a trademark and Impact is a trademark of Phenomenex. SCIEX and QTRAP are registered trademarks of AB SCIEX Pte. Ltd. AB SCIEX™ is being used under license.
FOR RESEARCH USE ONLY. Not for use in clinical diagnostic procedures.© 2020 Phenomenex, Inc. All rights reserved.
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