Erik Ruijters (Presenter)
MagnaMedics Diagnostics B.V.
Authorship: Erik Ruijters, Mario Wuttke
MagnaMedics Diagnostics B.V., Geleen, The Netherlands
| Short Abstract LC-MS/MS is replacing HPLC and immunoassays for immunosuppressant drug monitoring to achieve greater sensitivity and specificity. This study evaluated an in-line LC-MS/MS method where 10 µL blood samples collected by a microsampling device (Mitra) were prepared for analysis using a process (MagSi-TDMPREP) to eliminate interferences. Automation of this method resulted in a processing time of 30-45 minutes for 96 samples, robot dependent (4 or 8 span), while resulting in ≥ 80% recovery (RSD <15%) for four immunosuppressants. This method also included the tools required to handle, track, and prepare samples for direct LC-MS/MS injection or batching in vials/microtiter plates. |
Long Abstract
INTRODUCTION
Immunosuppressant drugs are routinely prescribed to transplant patients in order to suppress the body’s ability to reject the transplanted organs. The patient’s immunosuppressant drug therapy is closely monitored to obtain the optimum balance between therapeutic efficacy and the occurrence of adverse effects, which can prove difficult since they have a narrow therapeutic index and significant inter-individual variability in blood concentrations. To improve their monitoring practices, clinical labs are replacing HPLC and immunoassays with LC-MS/MS assays to achieve greater sensitivity and specificity. In addition, dried matrix microsampling is gaining attention as an alternative to the conventional wet sample collection for several compelling reasons. First, with improvements in LC-MS/MS technology, the blood volume requirements for bioanalysis make it unnecessary to collect such large volumes of blood. Second, dried microsampling, relative to wet sampling, offers a simplified collection, shipping, and sample preparation process. Third, it offers the option to send self-sampling kits to transplant patients at their home to reduce their number of visits to the lab for blood draws.
Here we present an in-line LC-MS/MS method for immunosuppressant drug monitoring that incorporates dried matrix microsampling with Mitra™ sampling tips (Neoteryx) and sample preparation using MagSi-TDMPREP (MagnaMedics) sample preparation process on an automated system. The Mitra microsampling tip rapidly wicks a fixed volume (e.g. 10 µL) of biological fluid. The internal volume of the microsampler is designed and precisely controlled to collect an accurate sample every time facilitating remote self-sampling. This sample can be shipped via regular mailing services, eliminating costly cold chain logistics, and analyzed in a hospital or service lab. The MagSi-TDMPREP sample preparation process is based on paramagnetic beads and eliminates interfering proteins, phospholipids and salts from biological samples prior to LC-MS/MS analysis. Because the precipitate is collected through magnetic separation, there is no need for centrifugation, making it easy to automate. Moreover, Solid Phase Extraction (SPE) clean-up or further concentration steps such as LLE or evaporation/reconstitution are not needed. The sample preparation method was implemented on a Hamilton Bioanalytical STARlet™ which has all the tools required to handle, track and prepare samples collected on a Mitra tip to be ready to inject direct into an LC-MS/MS system or batch-wise in HPLC vials or microtiter plates.
METHODS
Blood samples spiked with immunosuppressants (everolimus, sirolimus, 50 µg/L) were loaded on a Mitra Microsampler and allowed to dry overnight. The microsamplers were then loaded onto a Hamilton STARlet by using a prototype Mitra device holder to enable fully automated processing. The process started with a sample reconstitution to release the blood sample with immunosuppressants and change it back from a dried to a liquid form. The MagnaMedics reconstitution method is based on a buffer in combination with 20 µL of isotope labeled internal standard. The MagSi-TDMPREP sample preparation process was then performed, directly after the reconstitution, to clean the sample by removing proteins and unwanted components. In short: 40 μL MagSi-TDMPREP Type I bead solution was added to the reconstituted sample and mixed. Proteins were precipitated by addition of Organic Precipitation Reagent (OPR VI), followed by intense aspiration and dispensing of the mixture. After magnetic separation of the pellet, 80 μL of the supernatant was transferred to an HPLC vial. The extracts were then analyzed with a Shimadzu Nexera® X2 UHPLC combined with a LCMS-8050 Tandem Mass Spectrometer. A binary gradient of 2 mM ammonium formate + 0.1% formic acid in both water and methanol was used to separate 4 immunosuppressants and 4 isotopically labeled internal standards in 3.5 minutes on a Phenomenex Kinetex® C18 column (2.6μm, 2.1 x 50 mm ID).
RESULTS
Automation of this method resulted in a processing time of 30-45 minutes for 96 samples depending on the Hamilton STARlet robot used (4 or 8 span). Analysis of the immunosuppressants using Mitra microsampling to collect the blood sample coupled with MagnaMedics automated reconstitution and clean-up yielded good recovery (≥ 80%) and reproducibility (<15% RSD) for both immunosuppressants at the concentrations studied. By using multiple Mitra tips, the concentration of the sample could be increased making Mitra / MagSi-TDMPREP based assays also applicable for less sensitive LC-MS/MS systems or for diagnostic parameters which would otherwise require a concentration step because of the low therapeutic range.
CONCLUSIONS
Sample preparation on an Hamilton Bioanalytical STARlet provides an efficient way to perform TDM.
This study has shown that an in-line LC-MS/MS assay for immunosuppressant monitoring is possible with Mitra microsampling and the turnkey MagSi-TDMPREP sample preparation process. Clinical labs have the option to improve sensitivity and specificity for immunosuppressant monitoring with the added benefits of an automated, economical workflow, a reduced blood collection requirement, and a better patient experience with at-home sampling.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | MagnaMedics Diagnostics B.V. |
| 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 |