CPSA Shanghai 2012
From Bench to Decision Making - From Basics to Application
April 25 - 27, 2012
Renaissance Shanghai Pudong Hotel
Shanghai, China
Abstract
Friday
An Automated On-Line Card Extraction LC/MS System for the Determiantion of Clozpaine and its Metabolites in Rat Blood
Presenter:
Lester Taylor, Agilent Technologies, Inc., Santa Clara, CA, USA
Authors:
Lester Taylor, Na Pi Parra, Doug McIntyre, Yuqin Dai
Agilent Technologies, Inc., Santa Clara, CA, USA
Introduction
Dried blood spot (DBS) sampling provides several advantages when compared to conventional plasma sampling including use of small blood volumes, easy sample shipping and storage, and fewer biohazard concerns. When compared to traditional hole punching and off-line extraction methods, the on-line extraction LC/MS system enables automated flow through DBS analysis and reduces analysis time and manual preparation and increases sample throughput. This work describes the development and validation of an on-line card extraction DBS LC/MS method for the rapid quantitation of clozapine and its metabolites, norclozapine and clozapine-N-oxide, in rat blood. Excellent sensitivity, linearity, dynamic range, precision, accuracy, and reproducibility, were demonstrated. The quantitation performance of the method is comparable to that of an off-line sample extraction method. This approach could be readily applied to clozapine pharmacokinetics and metabolic profiling studies.
Method Development
The automated card extraction LC/MS system consists of a Prolab card extraction instrument for automated flow-through analysis of DBS cards, a binary pump for on-line sample extraction and cleaning, an isocratic pump for sample dilution, another binary pump for analytical LC separation, and an Agilent 6400 QQQ MS for analyte detection. The Prolab instrument is a CTC based autosampler which functions as master controller of the analytical setup to integrate with the pumps and the QQQ MS. Method development was performed in two phases to establish the optimum parameters for the on-line extraction and the analytical LC/MS detection. Clozapine and its metabolites were extracted from the DBS cards, mixed with internal standard, trapped on Trap1 column, and separated from matrix components using water and acetonitrile. After eluting from Trap1, the analytes were diluted with water to enable their retention on Trap2 column prior to the analytical run. Analytical separation was performed on a C18 column using 0.1% formic acid in water and acetonitrile. MRMs of m/z 327.0>192.1, 313.0>192.2, and 343.0>192.2, were employed for quantitation of clozapine, norclozapine, and clozapine-N-oxide, respectively. In this method, the restart for the next DBS extraction is performed from the extraction pump program while the analytical LC gradient is still running. Thus, the extraction overlaps the analytical run and achieves a cycle time of less than 10 min.
Results and Discussions
The limit of detection (LOD) in rat blood is 0.1 ng/mL for clozapine, norclozapine, and clozapine-N-oxide, and the lower limit of quantitation (LLOQ) is 0.5 ng/mL for all three analytes. Excellent accuracy (95-105%) and reproducibility (RSD<8.9%) were obtained at the LLOQ level. The calibration curves for all three analytes showed excellent linearity (R2>0.999) with over four orders of dynamic range (0.5-10000 ng/mL). The method accuracy, reproducibility, and precision were evaluated at eleven standard concentrations. The accuracy of measurement was determined to be 85–109% for all analytes. The triplicate reproducibility is excellent with RSD <6.2% for all analytes. The precision (RSD) of analyses over the calibration dynamic range is <8.9% for all analytes.
The quantitation performance of the on-line extraction method was compared to that of a hole punching manual extraction method and comparable results were observed. The off-line method delivered a linear dynamic range of 0.5-10000 ng/mL (R2>0.999) with an LLOQ level of 0.5 ng/mL. The accuracy, reproducibility and precision of the off-line method were determined to be 88-108%, <9.0% and <6.6%, respectively. The comparable quantitation performance demonstrated the validity of the automated card extraction LC/MS system for direct analysis of DBS cards for rapid pharmacokinetics studies and metabolite profiling of clozapine.
