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Abstract Introduction
Immunosuppressant drug monitoring is commonly utilized in organ donation rejection prevention and in organ cancers such as kidney, pancreatic, and epithelial cell cancers. The continuous monitoring of these drugs saves lives, therefore a quick and easy sample preparation method prior to analysis is imperative in the hospital setting. Unfortunately, immunosuppressants are highly protein bound, and recoveries with traditional methods can be scarce and lack sensitivity. The accurate and robust filtration of four immunosuppressants (Cyclosporin A, Everolimus, Sirolimus, and Tacrolimus) are demonstrated in this poster.
Objectives
This method is quick, easy, and can be performed by anyone looking to analyze immunosuppressants in whole blood; this method provides clean extracts and therefore less instrument maintenance.
Methods
A MICROLAB® NIMBUS96 automated liquid handler (referred to as ALH) is utilized for sample preparation, however this can be performed manually or with other automated pipetting platforms. First, 50 µL of 0.2 M ZnSO4 in water is added to 50 µL of whole blood in a well plate for the initial step of protein precipitation. The solution is mixed thoroughly 10 times and allowed to settle for several seconds. Next, a 150 µL aliquot of acetonitrile (ACN) is added and the solution is mixed 20 times thoroughly for a fully precipitated sample. The ALH takes a fresh set of tips, aspirates the sample solution, goes Tip-on-Tip with the filtration tips, moves to a new well plate location, and dispenses the sample through the filtration tip into a well plate. The ALH disposes of the tips and picks up another set of tips. The tips transfer 100 µL of acetonitrile and 25 µL of HPLC water to the final well plate for injection. The tips then transfer a 125 µL aliquot of the filtrate to the injection well plate, mixing the solution to ensure homogenous composition for injection. The entire method from start to finish was 10 minutes.
Analysis was performed on a Shimadzu LC40 paired with a SCIEX 6500+ tandem mass spectrometer (LC-MS/MS). The analytical LC column is a Restek Force Biphenyl, 3 µm, 50 x 2.1 mm LC Column (PN 9629352) paired with a Restek Force Biphenyl, 5 x 2.1 mm EXP Guard Cartridge (PN 962950252). The choice of a biphenyl column allows for optimal separation and more flexibility in sample composition prior to injection; a 5 µL injection volume is used and the LC method is 8 minutes. The column is heated to 70°C, and the mobile phases are [A] 0.1% formic acid in HPLC water and [B] 100% methanol. The internal standards used are Everolimus D4 (Cerilliant, TX) and Cyclosporin A 11N15 (Cerilliant, TX).
Results
The immunosuppressants LOQs (level of quantitation) are determined to be 1 ng/mL for Tacrolimus, Sirolimus, and Everolimus and 10 ng/mL for Cyclosporin A. Reference ranges, based on available literature, for these analytes are above the determined LOQs. During a linearity study conducted in correlation with 2 UTAK Control QC’s, accuracy values were found between 88.5% and 106.6% for all compounds. The recovery is found to be 93% to 100% for all analytes from DPX filtration and a total recovery from filtration and protein precipitation is found to be 72% to 83%, where most of the recovery loss is from the protein precipitation.
Conclusion
For highly protein bound analytes such as immunosuppressants in whole blood, an effective sample preparation protocol is necessary to ensure accuracy of patient results. A fast and reproducible method for the analysis of immunosuppressants in whole blood is needed to determine how well a patient has acclimated to a new organ transplant. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
