Limian Zhao
Agilent Technologies
Bio: Sr. application scientist focusing on sample preparation techniques, methodologies, and applications.
Authorship: Limian Zhao and Joni Stevens
Agilent Technologies
| Short Abstract The Captiva Enhanced Matrix Removal-Lipid (EMR-Lipid) is a pass-through lipid cleanup product implemented in a convenient 96-well plate or SPE cartridge format. This study demonstrates an analytical method for analysis of 24 abuse drugs in whole blood using in-well protein precipitation (PPT) followed with Captiva EMR-Lipid cleanup by LC/MS/MS. Samples were prepared using in-well PPT for protein removal, followed by Captiva EMR-Lipid cleanup for lipids removal. The entire study was conducted in the 96-well plate as a batch process with minimal sample transfer and automatable operations. The analytical method provided >99% removal of phospholipids from whole blood, and an average of 80% absolute recoveries for target analytes. The analytical method was verified for the calibration range of 0.1(0.5) – 20 ng/mL in whole blood with superior accuracy and precision. |
Long Abstract
Introduction
Determination of abuse drugs (DoA) in whole blood has been considered as the first choice for the quantitative analysis of abuse drugs in forensic labs. Therefore, the solid quantitative method is critical to achieve accurate and reliable results. Biological sample matrix like whole blood is very complex containing endogenous and exogenous substances. It is also quite viscous and difficult to handle. Therefore, appropriate sample preparation is critical to extract target analytes, clean unwanted matrix interferences, and also simplify the sample operation. In addition, the 96-well plated based sample preparation has been widely used for high-throughput bioanalysis for quantitative determination of target analytes in biological matrices, due to its batch process and automatable operations, and also limited sample volume used for biological sample analysis.
Captiva EMR-Lipid plate provides pass-through matrix cleanup to remove major lipid substances from sample matrix without unwanted analyte loss. By selectively interaction with the long aliphatic chain of the lipid compounds, the EMR-Lipid cleanup provides efficient phospholipids and other classes of lipids from biological fluids after protein precipitation (PPT). The highly selective interaction mechanism also assures minimal retention of target analytes during cleanup. In addition, the in-well protein precipitation reduces the sample transfer and allows automatic operation. All of above features make Captiva EMR-Lipid cleanup an excellent option to prepare samples for DoA analysis in whole blood in forensic studies.
Methods
A quantitative analytical determination of 24 abuse drug compounds in whole blood was developed and verified. The sample preparation includes in-well protein precipitation using cold 95:5 ACN/MeOH solvent to remove proteins firstly, followed with pass-through lipids removal using Captiva EMR-Lipid 96-well plate. The matrix removal efficiency was evaluated by total phospholipids monitoring with and without EMR-Lipid cleanup. The matrix ion suppression on the target analytes was evaluated qualitatively and quantitatively. The analyte absolute recovery was assess in both low and high spiking levels in whole blood. The analytical method was then verified through three-day accuracy and precision runs. The quantitation results were also compared between with and without the use of EMR-Lipid cleanup.
Results
The results showed that EMR-Lipid cleanup provided >99% of phospholipids removal and greatly reduced matrix ion suppression. An average of 80% absolute recovery for the target analytes at both low and high spiking level indicated minimal demonstrated the minimal impact on target analytes by EMR-Lipid cleanup. The analytical method verification showed excellent calibration curve linearity with R2 > 0.99 with limit of quantitation down to 0.1 ng/mL in whole blood, exceptional accuracy for all five levels of QCs (<15%) and precision (RSD <15%) for both intra- and inter-day runs. Due to the significantly cleaner sample matrix, the column contamination and carryover issues caused by matrix phospholipids were greatly improved, which allowed the use of shorter LC gradient, increased the column lifetime and reduced instrument downtime for system cleanup.
Conclusions & Discussion
A simple sample preparation analytical method using PPT followed by Captiva EMR-Lipid cleanup was verified for the high-throughput quantitative determination of 24 abuse drug compounds in human whole blood. The analytical method provides superior dynamic range and calibration linearity, delivers exceptional intra- and inter-day accuracy and precision. The pass-through Captiva EMR-Lipid cleanup provide >99% of phospholipids removal, and therefore reduce matrix ion suppression effect.
For Research Use Only. Not for use in diagnostic procedures.
References
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