Daisuke Kawakami (Presenter)
Shimadzu Corporation
Authorship: Daisuke Kawakami (1, 3): Taku Tsukamoto (1, 3): Naohiro Ohara (3): Kaori Shinmyozu (3): Yuko Shimamoto (3): Hikaru Shibata (1, 2): Brian Field (2): Yoshihiro Hayakawa (1): Takafumi Tanigaki (1): Shin Nakamura (1): Takeshi Kuwahara (3):
(1) Shimadzu Corporation, Kyoto, Japan (2)Shimadzu Scientific Instruments, Inc, Maryland, U.S.A. (3) National Cerebral and Cardiovascular Center, Osaka, Japan
| Short Abstract LC-MS/MS has become essential tool for monitoring the concentration of drugs in biological samples due to its high level of sensitivity and specificity; however, manual sample preparation often involves several complicated manual steps which can introduce error into the results. In this study, we investigated the ability to analyze for Novel Oral Anticoagulants by LC-MS/MS using automated sample preparation to process large sample sets. We validated the automated method by comparing the data collected on the automated system to the data collected using a manual sample preparation protocol. |
Long Abstract
Introduction
LC-MS/MS has become essential tool for monitoring the concentration of drugs in biological samples due to its high level of sensitivity and specificity; however, manual sample preparation often involves several complicated manual steps which can introduce error into the results. Additionally, the time consuming nature of the sample preparation and the large number of samples makes LC-MS/MS a less desirable method. Automated sample preparation has been shown to eliminate human error, as well as increase laboratory efficiency, making LC-MS/MS a feasible method to incorporate in the clinic.
In this study, we investigated the ability to analyze for Novel Oral Anticoagulants (NOACs) by LC-MS/MS (LCMS-8040, Shimadzu) using automated sample preparation (CLAM-2000, Shimadzu) to process large sample sets. The CLAM-2000 has the ability to perform a variety of steps appropriate for automated sample preparation by LC-MS/MS including precipitation, filtration, heating, shaking, and pipetting. This system is seamlessly integrated with the LCMS system requiring no human involvement after loading the biological samples into the sample chamber. We validated the automated method by comparing the data collected on the automated system to the data collected using a manual sample preparation protocol. An additional consideration we evaluated was the number of human hours dedicated towards preparing samples for this assay using both methods and the ability to incorporate a STAT sample into the queue with rapid turn-around time for results.
Methods
Plasma or plasma spiked with NOAC was loaded directly into the CLAM-2000 for sample processing. The CLAM-2000 was programmed to perform protein precipitation using acetonitrile followed by filtration and sample collection. The sample is then transported using an arm from the CLAM-2000 to the HPLC for LC-MS/MS analysis and no human intervention was required. For the manual sample preparation, acetonitrile precipitation followed by centrifugation was performed. After the sample preparation the automated and manually prepared samples were analyzed using the same method. 2ƒÊl of sample were injected and separated by HPLC over a C18 column (Mastro C18 100mm X 2mm 3.0ƒÊm , Shimadzu GLC) at 50‹C with a binary gradient system of water with 0.1% formic acid and methanol with 0.1% formic acid at a flow rate of 0.4 ml/min.
Results
Plasma spiked with four NOAC (Apixaban, Rivaroxaban, Edoxaban, Dabigatran) were used for calibration. Each of the compounds were calibrated within the range from 5ng/mL to 500ng/mL by the six calibration points (5, 10, 20, 50, 200, 500ng/mL). The calibration curves showed good linearity (R^2>0.992). The reproducibility (N=6) at six concentrations, including LLOQ, of each compounds was excellent (CV<10%). Different day reproducibility (N=6) for 5 days at three concentrations (5, 50, 500ng/mL) as well (CV<15%). Comparison of concentration between manual sample preparation and automated sample preparation using plasma from patients who have NOAC medication shows good agreement. The difference of the automated operation against the manual operation was between -15% and 15%.
In the routine work observation, we found that the work time of the technician for calibration, ten samples and the addition of a STAT sample was reduced from 60 minutes to 10 minutes by the automated system. We also obtained a STAT sample result within twenty minutes after setting the sample into the system.
Conclusions
We completed NOAC analysis using a LC-MS/MS coupled to an automated sample preparation system. The results shows the capability of the system for large sample set analyses with improved accuracy and precision. We also found that the system has the additional benefits of flexible configuration and programming, including incorporation of STAT samples, as well as the elimination of human error associated with manual sample handling.
References & Acknowledgements:
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