Hikaru Shibata (Presenter)
Shimadzu Scientific Instruments Inc.
Authorship: Hikaru Shibata 1, 2: Taku Tsukamoto 1, 3: Daisuke Kawakami 1, 3: Tomoko Inagaki 1: Naohiro Ohara 3: Kaori Shinmyozu 3: Yuko Shimamoto 3: 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 The limits of traditional analytical methods, such as LC-MS/MS, are progressively being pushed further with regard to robustness, ease of use, sensitivity, throughput, cost effectiveness and accuracy. Here we propose a novel approach to automated sample preparation by combining an on-line automated sample preparation instrument (CLAM-2000, Shimadzu) for LC-MS/MS analysis (LCMS-8040, Shimadzu). In this study, we investigated ability to analyze for antiepileptic drugs and antiarrhythmic drugs using the CLAM-2000 for automated sample preparation for LC-MS/MS. We validated feasibility of this system by measuring precision and accuracy of the drugs which have various chemical properties. |
Long Abstract
Introduction
The limits of traditional analytical methods, such as LC-MS/MS, are progressively being pushed further with regard to robustness, ease of use, sensitivity, throughput, cost effectiveness and accuracy. This is a result of common LC-MS/MS methods being adapted for large scale research studies involving hundreds to thousands of complex biological samples. The effectiveness of the analysis typically depends on time consuming, user dependent methods in order to simplify the sample matrix to reliably target specific analytes.
Lab automation offers the ability to remove user errors while processing large sample sets with very little user involvement. Two options for lab automation are robotic liquid handling systems and on-line systems. While robotic systems offer the ability to process many samples at the same time, setup and troubleshooting can be extremely time-consuming. These also require users to manually transfer samples from the robot to the HPLC for LC-MS/MS analysis. On-line sample preparation machines, on the other hand, process samples sequentially, in parallel (multiple steps being performed simultaneously) and seamlessly integrate with the LC-MS/MS system, thus requiring no human intervention after loading the biological sample.
Here we propose a novel approach to automated sample preparation by combining an on-line automated sample preparation instrument (CLAM-2000, Shimadzu) for LC-MS/MS analysis (LCMS-8040, Shimadzu). The CLAM-2000 automates common steps in laboratory protocols such as precipitation, filtration, shaking, heating and pipetting. The sample is delivered directly into the autosampler of the High Performance Liquid Chromatography (HPLC) system prior to quantitation by multiple reaction monitoring (MRM). Steps can be performed in parallel to accelerate throughput.
In this study, we investigated ability to analyze for antiepileptic drugs and antiarrhythmic drugs using the CLAM-2000 for automated sample preparation for LC-MS/MS. We validated feasibility of this system by measuring precision and accuracy of the drugs which have various chemical properties.
Antiepileptic Drug Analysis
Serum spiked with six antiepileptic drugs and a metabolite was analyzed using automated on-line sample preparation combined with LC-MRM. The sample preparation involved steps where samples were precipitated with acetonitrile and filtrated by CLAM-2000. Using automated sample preparation and parallel processing, the total analysis time per sample was 9 minutes. For quantitative validation, we obtained satisfied linearity (R2>0.99) within the appropriate ranges of each compounds. Accuracy of all compounds was within the range from 115% to 85% of each calibration points, including LLOQ. And reproducibility (N=6) was also excellent (CV < 15%).
Antiarrhythmic drugs
We focused on hydrophilicity of chemical properties to validate the feasibility of the system. Amiodarone is an antiarrhythmic drug. This compound shows strong hydrophobicity (The partition coefficient; Log P = 6.9326). On the other hand, Sotalol is an antiarrhythmic drug which has strong hydrophilicity (Log P = 2.6342). We analyzed these two drugs and N-desethylamiodarone, a metabolite of amiodarone, by the system. These results compared to the results obtained with the antiepileptic drugs.
Conclusion
We completed antiepileptic drugs and antiarrhythmic drugs 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 regardless of hydrophilicity of the chemicals. The accuracy and reproducibility values illustrate the great value in a fully integrated and automated system for LC-MS/MS analysis.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Shimadzu |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |